Plant proteinases

ABSTRACT

This invention relates to an isolated nucleic acid fragment encoding a proteinase. The invention also relates to the construction of a chimeric gene encoding all or a portion of the proteinase, in sense or antisense orientation, wherein expression of the chimeric gene results in production of altered levels of the proteinase in a transformed host cell.

[0001] This application claims the benefit of U.S. Provisional Application No. 60/119,599, filed Feb. 10, 1999.

FIELD OF THE INVENTION

[0002] This invention is in the field of plant molecular biology. More specifically, this invention pertains to nucleic acid fragments encoding proteinases in plants and seeds.

BACKGROUND OF THE INVENTION

[0003] The protein turnover in all organisms must be highly regulated since proteins within the same subcellular compartment have very different half-lives. The ubiquitin system is the major energy-dependent protease in the cytosol where ubiquitin binds to the protein to be degraded and then the multisubunit proteasome processes it. Another energy-dependent protease is the membrane-bound ATP-dependent proteinase or Clp. Other cellular proteinases are the calcium-dependent calpain, cysteine proteases, metallo-peptidases and other serine-type peptidases.

[0004] Calpain is an intracellular calcium-dependent protease activated at cell membranes which cleaves cytoskeletal and submembranous proteins and is probably involved in the calcium-dependent regulation of the cytoskeletal reorganization. The proteolytic activity of calpain and the binding of calpain to membranes is inhibited by the endogenous inhibitor calpastatin. Mammalian and insect tissues each has at least two different calpain isozymes. Each isozyme contains a different large subunit and an identical small subunit. A third calpain isozyme containing only a large subunit called p94 has been identified in mammalian tissues. All three large subunits contain four conserved domains including a cysteine protease domain and a calcium-binding domain. The p94 large subunit contains three additional domains probably involved in protease activity regulation and/or intracellular localization (Sorimachi et al. (1989) J. Biol. Chem. 264:20106-20111). While no calpain activity has been detected in chromatographic extracts from Elodea densa tissues (Wolfe et al (1989) Life Sci. 45:2093-2101), the p94 large subunit may be present at a lower concentration than the sensitivity of their assay (1 microgram per 0.25 g) and thus may be found in plant tissues.

[0005] Two different cysteine proteinases have been isolated from tissues of several species including arabidopsis, pea, rice, barley and corn. The barley cysteine proteinases EP-A and EP-B are induced by the presence of gibberellic acid and play a central role in the breakdown of endosperm storage proteins (hordeins) in the aleurone layers (Koehler and Ho (1988). Plant Physiol. 87:95-103). These cysteine proteinases are members of a small gene family composed of four to five different genes and are translated as prosequences which follow a post-translationally multistep processing to mature products. The barley cysteine proteinases are differentially hormonally induced and temporally regulated (Koehler and Ho (1990) Plant Cell 2:769-783). Cysteine proteinases isolated from corn seeds are differentially expressed and have different subcellular localizations (Domoto et al. (1995) Biochim. Biopys. Acta 1263:241-244).

[0006] The CLP system was first identified in E. coli and later in plant chloroplasts. This proteolytic activity is induced by heat shock, by salt or oxidative stress or by glucose or oxygen limitation. Two different components are present in the CLP system, a protease and an ATP-binding factor. Two different types of ATP-binding factors, CLPA and CLPX, present specific substrates to the protease domain to facilitate their degradation. A six-membered ring formed by CLPX subunits binds to ATP and to two seven-member rings of CLPP to produce the active enzyme. This complex is structurally analogous to the one formed by CLPP and CLPA (Grimaud et al. (1998) J. Biol. Chem. 273:12476-12481).

SUMMARY OF THE INVENTION

[0007] The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 40 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a calpain p94 polypeptide of SEQ ID NOs:2, 4, 6, 30, 32, and 34. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

[0008] The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 150 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a cysteine protease 1 polypeptide of SEQ ID NOs:8, 10, 36, and 38. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

[0009] The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 200 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a cysteine protease 2 polypeptide of SEQ ID NOs:12 and 40. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

[0010] The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a polypeptide of at least 175 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of a CLPA polypeptide of SEQ ID NOs:14, 16, 18, 42, 44, and 46. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

[0011] The present invention relates to isolated polynucleotides comprising a nucleotide sequence encoding a CLPP polypeptide selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 48, 50, 52, 54, and 56. The present invention also relates to an isolated polynucleotide comprising the complement of the nucleotide sequences described above.

[0012] It is preferred that the isolated polynucleotides of the claimed invention consist of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and 56. The present invention also relates to an isolated polynucleotide comprising a nucleotide sequences of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences.

[0013] The present invention relates to a chimeric gene comprising an isolated polynucleotide of the present invention operably linked to suitable regulatory sequences.

[0014] The present invention relates to an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention. The host cell may be eukaryotic, such as a yeast or a plant cell, or prokaryotic, such as a bacterial cell. The present invention also relates to a virus, preferably a baculovirus, comprising an isolated polynucleotide of the present invention or a chimeric gene of the present invention.

[0015] The present invention relates to a process for producing an isolated host cell comprising a chimeric gene of the present invention or an isolated polynucleotide of the present invention, the process comprising either transforming or transfecting an isolated compatible host cell with a chimeric gene or isolated polynucleotide of the present invention.

[0016] The present invention relates to a calpain p94 polypeptide of at least 40 amino acids comprising at least 85% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 30, 32, and 34.

[0017] The present invention relates to a cysteine protease 1 polypeptide of at least 150 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 10, 36, and 38.

[0018] The present invention relates to a cysteine protease 2 polypeptide of at least 200 amino acids comprising at least 80% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:12 and 40.

[0019] The present invention relates to a CLPA polypeptide of at least 175 amino acids comprising at least 95% homology based on the Clustal method of alignment compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 16, 18, 42, 44, and 46.

[0020] The present invention relates to a CLPP polypeptide selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 48, 50, 52, 54, and 56.

[0021] The present invention relates to a method of selecting an isolated polynucleotide that affects the level of expression of a proteinase polypeptide in a host cell, preferably a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; (b) introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; (c) measuring the level the cysteine protease I, the cysteine protase 2, the calpain large subunit, the CLP protease proteolytic subunit or the CLP protease ATP binding subunit polypeptide in the host cell containing the isolated polynucleotide; and (d) comparing the level of the cysteine protease I, the cysteine protase 2, the calpain large subunit, the CLP protease proteolytic subunit or the CLP protease ATP binding subunit polypeptide in the host cell containing the isolated polynucleotide with the level of the cysteine protease I, the cysteine protase 2, the calpain large subunit, the CLP protease proteolytic subunit or the CLP protease ATP binding subunit polypeptide in the host cell that does not contain the isolated polynucleotide.

[0022] The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a cysteine protease I, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit polypeptide, preferably a plant cysteine protease I, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit polypeptide, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a cysteine protease I, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit amino acid sequence.

[0023] The present invention also relates to a method of obtaining a nucleic acid fragment encoding all or a substantial portion of the amino acid sequence encoding a cysteine protease I, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit polypeptide comprising the steps of: probing a cDNA or genomic library with an isolated polynucleotide of the present invention; identifying a DNA clone that hybridizes with an isolated polynucleotide of the present invention; isolating the identified DNA clone; and sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.

[0024] The present invention relates to a composition, such as a hybridization mixture, comprising an isolated polynucleotide of the present invention.

[0025] The present invention relates to an isolated polynucleotide of the present invention comprising at least one of 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55.

[0026] The present invention relates to an expression cassette comprising an isolated polynucleotide of the present invention operably linked to a promoter.

[0027] The present invention relates to a method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of the present invention or an expression cassette of the present invention; and (b) growing the transformed host cell, preferably plant cell, such as a monocot or a dicot, under conditions which allow expression of the cysteine protease I, the cysteine protase 2, the calpain large subunit, the CLP protease proteolytic subunit or the CLP protease ATP binding subunit polynucleotide in an amount sufficient to complement a null mutant to provide a positive selection means.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

[0028] The invention can be more fully understood from the following detailed description and the accompanying Sequence Listing which form a part of this application.

[0029] Table 1 lists the polypeptides that are described herein, the designation of the cDNA clones that comprise the nucleic acid fragments encoding polypeptides representing all or a substantial portion of these polypeptides, and the corresponding identifier (SEQ ID NO:) as used in the attached Sequence Listing. The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. TABLE 1 PLANT PROTEINASES SEQ ID NO: Protein Clone Designation (Nucleotide) (Amino Acid) Corn Calpain p94 Subunit cbn2.pk0039.c2 1 2 Rice Calpain p94 Subunit rs11n.pk013.h14 3 4 Soybean Calpain p94 Subunit ses9c.pk001.j23 5 6 Rice Cysteine Protease 1 rr1.pk084.j16 7 8 Wheat Cysteine Protease 1 Contig of: 9 10 wdk1c.pk009.j19 wre1n.pk164.b11 Soybean Cysteine Protease 2 Contig of: 11 12 sgs2c.pk002.p14 srr3c.pk003.d10 scb1c.pk003.d8 Corn CLP ATP Binding Subunit p0110.cgsmk69r 13 14 Rice CLP ATP Binding Subunit Contig of: 15 16 r1r6.pk0083.f9 r1r24.pk0088.f7 r1r6.pk0029.d7 Wheat CLP ATP Binding Subunit w1m96.pk032.n8 17 18 Corn CLP Proteolytic Subunit p0060.coran66r 19 20 Rice CLP Proteolytic Subunit rsr9n.pk004.p5 21 22 Soybean CLP Proteolytic Subunit scb1c.pk004.k24 23 24 Wheat CLP Proteolytic Subunit w1e1n.pk0042.f7 25 26 Wheat CLP Proteolytic Subunit w1k8.pk0006.a4 27 28 Corn Calpain p94 Subunit cbn2.pk0039.c2:fis 29 30 Rice Calpain p94 Subunit rs11n.pk013.h14:fis 31 32 Soybean Calpain p94 Subunit ses9c.pk001.j23:fis 33 34 Rice Cysteine Protease 1 rr1.pk084.j16:fis 35 36 Wheat Cysteine Protease 1 wdk1c.pk009.j19:fis 37 38 Soybean Cysteine Protease 2 srr3c.pk003.d10:fis 39 40 Corn CLP ATP Binding Subunit p0110.cgsmk69r:fis 41 42 Rice CLP ATP Binding Subunit r1r24.pk0088.f7:fis 43 44 Wheat CLP ATP Binding Subunit w1m96.pk032.n8:fis 45 46 Corn CLP Proteolytic Subunit p0060.coran66r:fis 47 48 Rice CLP Proteolytic Subunit rsr9n.pk004.p5:fis 49 50 Soybean CLP Proteolytic Subunit scb1c.pk004.k24:fis 51 52 Wheat CLP Proteolytic Subunit w1e1n.pk0042.f7:fis 53 54 Wheat CLP Proteolytic Subunit w1k8.pk0006.a4:fis 55 56

[0030] The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

DETAILED DESCRIPTION OF THE INVENTION

[0031] In the context of this disclosure, a number of terms shall be utilized. As used herein, a “polynucleotide” is a nucleotide sequence such as a nucleic acid fragment. A polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded, that optionally contains synthetic, non-natural or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof. An isolated polynucleotide of the present invention may include at least one of 60 contiguous nucleotides, preferably at least one of 40 contiguous nucleotides, most preferably one of at least 30 contiguous nucleotides derived from SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55, or the complement of such sequences.

[0032] As used herein, “contig” refers to a nucleotide sequence that is assembled from two or more constituent nucleotide sequences that share common or overlapping regions of sequence homology. For example, the nucleotide sequences of two or more nucleic acid fragments can be compared and aligned in order to identify common or overlapping sequences. Where common or overlapping sequences exist between two or more nucleic acid fragments, the sequences (and thus their corresponding nucleic acid fragments) can be assembled into a single contiguous nucleotide sequence.

[0033] As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by gene silencing through for example antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-à-vis the ability to mediate gene silencing or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.

[0034] Substantially similar nucleic acid fragments may be selected by screening nucleic acid fragments representing subfragments or modifications of the nucleic acid fragments of the instant invention, wherein one or more nucleotides are substituted, deleted and/or inserted, for their ability to affect the level of the polypeptide encoded by the unmodified nucleic acid fragment in a plant or plant cell. For example, a substantially similar nucleic acid fragment representing at least one of 30 contiguous nucleotides derived from the instant nucleic acid fragment can be constructed and introduced into a plant or plant cell. The level of the polypeptide encoded by the unmodified nucleic acid fragment present in a plant or plant cell exposed to the substantially similar nucleic fragment can then be compared to the level of the polypeptide in a plant or plant cell that is not exposed to the substantially similar nucleic acid fragment.

[0035] For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a nucleic acid fragment which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded polypeptide, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the polypeptide molecule would also not be expected to alter the activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Consequently, an isolated polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences may be used in methods of selecting an isolated polynucleotide that affects the expression of a proteinase polypeptide in a host cell. A method of selecting an isolated polynucleotide that affects the level of expression of a polypeptide in a host cell (eukaryotic, such as plant or yeast, prokaryotic such as bacterial, or viral) may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.

[0036] Moreover, substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (Hames and Higgins, Eds. (1985) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms. Post-hybridization washes determine stringency conditions. One set of preferred conditions uses a series of washes starting with 6X SSC, 0.5% SDS at room temperature for 15 min, then repeated with 2X SSC, 0.5% SDS at 45° C. for 30 min, and then repeated twice with 0.2X SSC, 0.5% SDS at 50° C. for 30 min. A more preferred set of stringent conditions uses higher temperatures in which the washes are identical to those above except for the temperature of the final two 30 min washes in 0.2X SSC, 0.5% SDS was increased to 60° C. Another preferred set of highly stringent conditions uses two final washes in 0.1X SSC, 0.1% SDS at 65° C.

[0037] Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art. Suitable nucleic acid fragments (isolated polynucleotides of the present invention) encode polypeptides that are at least about 70% identical, preferably at least about 80% identical to the amino acid sequences reported herein. Preferred nucleic acid fragments encode amino acid sequences that are about 85% identical to the amino acid sequences reported herein. More preferred nucleic acid fragments encode amino acid sequences that are at least about 90% identical to the amino acid sequences reported herein. Most preferred are nucleic acid fragments that encode amino acid sequences that are at least about 95% identical to the amino acid sequences reported herein. Suitable nucleic acid fragments not only have the above homologies but typically encode a polypeptide having at least 50 amino acids, preferably at least 100 amino acids, more preferably at least 150 amino acids, still more preferably at least 200 amino acids, and most preferably at least 250 amino acids. Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW5 and DIAGONALS SAVED=5.

[0038] A “substantial portion” of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises. Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. BioL 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/). In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene-specific oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

[0039] “Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly, the instant invention relates to any nucleic acid fragment comprising a nucleotide sequence that encodes all or a substantial portion of the amino acid sequences set forth herein. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a nucleic acid fragment for improved expression in a host cell, it is desirable to design the nucleic acid fragment such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0040] “Synthetic nucleic acid fragments” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form larger nucleic acid fragments which may then be enzymatically assembled to construct the entire desired nucleic acid fragment. “Chemically synthesized”, as related to nucleic acid fragment, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of nucleic acid fragments may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the nucleic acid fragments can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

[0041] “Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

[0042] “Coding sequence” refers to a nucleotide sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

[0043] “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0044] The “translation leader sequence” refers to a nucleotide sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner and Foster (1995) Mol. Biotechnol. 3:225-236).

[0045] The “3′ non-coding sequences” refer to nucleotide sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al. (1989) Plant Cell 1:671-680.

[0046] “RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into polypeptide by the cell. “cDNA” refers to a double-stranded DNA that is complementary to and derived from mRNA. “Sense” RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell. “Antisense RNA” refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (see U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

[0047] The term “operably linked” refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

[0048] The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

[0049] “Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

[0050] “Mature” protein refers to a post-translationally processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

[0051] A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys. 100:1627-1632).

[0052] “Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

[0053] Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook et al. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

[0054] Nucleic acid fragments encoding at least a portion of several proteinases have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous proteins from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

[0055] For example, genes encoding other cysteine protease 1s, cysteine protase 2s, calpain p94s, CLPAs, or CLPPs, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate fall length cDNA or genomic fragments under conditions of appropriate stringency.

[0056] In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al. (1988) Proc. Natl. Acad. Sci. USA 85:8998-9002) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al. (1989) Proc. Natl. Acad. Sci. USA 86:5673-5677; Loh et al. (1989) Science 243:217-220). Products generated by the 3′ and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman and Martin (1989) Techniques 1:165). Consequently, a polynucleotide comprising a nucleotide sequence of at least one of 60 (preferably one of at least 40, most preferably one of at least 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences may be used in such methods to obtain a nucleic acid fragment encoding a substantial portion of an amino acid sequence of a polypeptide. The present invention relates to a method of obtaining a nucleic acid fragment encoding a substantial portion of a proteinase polypeptide (such as a cysteine protease 1, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit) preferably a substantial portion of a plant polypeptide of a gene, comprising the steps of: synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 60 (preferably at least one of 40, most preferably at least one of 30) contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55, and the complement of such nucleotide sequences; and amplifying a nucleic acid fragment (preferably a cDNA inserted in a cloning vector) using the oligonucleotide primer. The amplified nucleic acid fragment preferably will encode a portion of a cysteine protease I, a cysteine protase 2, a calpain large subunit, a CLP protease proteolytic subunit or a CLP protease ATP binding subunit polypeptide.

[0057] Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner (1984) Adv. Immunol 36:1-34; Maniatis).

[0058] The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed polypeptides are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of protein stability in those cells. The cysteine proteinases or the calpain p94 subunit are useful for the study and control of apoptosis in plants since they are induced by stress. Manipulating the expression of proteinases in plant cells will be useful for controlling cell death (apoptosis) caused by disease. Manipulation of the expression of proteinases in tissue culture will improve the survival rate during the harsh transformation treatments and during maintenance in tissue culture.

[0059] Overexpression of the proteins of the instant invention may be accomplished by first constructing a chimeric gene in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric gene may also comprise one or more introns in order to facilitate gene expression.

[0060] Plasmid vectors comprising the isolated polynucleotide (or chimeric gene) may be constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al. (1985) EMBO J. 4:2411-2418; De Almeida et al. (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

[0061] For some applications it may be useful to direct the instant polypeptides to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by directing the coding sequence to encode the instant polypeptides with appropriate intracellular targeting sequences such as transit sequences (Keegstra (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization (Chrispeels (1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53), or nuclear localization signals (Raikhel (1992) Plant Phys. 100:1627-1632) with or without removing targeting sequences that are already present. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of use may be discovered in the future.

[0062] It may also be desirable to reduce or eliminate expression of genes encoding the instant polypeptides in plants for some applications. In order to accomplish this, a chimeric gene designed for co-suppression of the instant polypeptide can be constructed by linking a gene or gene fragment encoding that polypeptide to plant promoter sequences. Alternatively, a chimeric gene designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the gene or gene fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric genes could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

[0063] Molecular genetic solutions to the generation of plants with altered gene expression have a decided advantage over more traditional plant breeding approaches. Changes in plant phenotypes can be produced by specifically inhibiting expression of one or more genes by antisense inhibition or cosuppression (U.S. Pat. Nos. 5,190,931, 5,107,065 and 5,283,323). An antisense or cosuppression construct would act as a dominant negative regulator of gene activity. While conventional mutations can yield negative regulation of gene activity these effects are most likely recessive. The dominant negative regulation available with a transgenic approach may be advantageous from a breeding perspective. In addition, the ability to restrict the expression of specific phenotype to the reproductive tissues of the plant by the use of tissue specific promoters may confer agronomic advantages relative to conventional mutations which may have an effect in all tissues in which a mutant gene is ordinarily expressed.

[0064] The person skilled in the art will know that special considerations are associated with the use of antisense or cosuppression technologies in order to reduce expression of particular genes. For example, the proper level of expression of sense or antisense genes may require the use of different chimeric genes utilizing different regulatory elements known to the skilled artisan. Once transgenic plants are obtained by one of the methods described above, it will be necessary to screen individual transgenics for those that most effectively display the desired phenotype. Accordingly, the skilled artisan will develop methods for screening large numbers of transformants. The nature of these screens will generally be chosen on practical grounds. For example, one can screen by looking for changes in gene expression by using antibodies specific for the protein encoded by the gene being suppressed, or one could establish assays that specifically measure enzyme activity. A preferred method will be one which allows large numbers of samples to be processed rapidly, since it will be expected that a large number of transformants will be negative for the desired phenotype.

[0065] The instant polypeptides (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting the polypeptides of the instant invention in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant polypeptides are microbial hosts. Microbial expression systems and expression vectors containing regulatory sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant polypeptides. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded proteinases. An example of a vector for high level expression of the instant polypeptides in a bacterial host is provided (Example 10).

[0066] All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J Hum. Genet. 32:314-331).

[0067] The production and use of plant gene-derived probes for use in genetic mapping is described in Bernatzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

[0068] Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Nonmammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).

[0069] In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

[0070] A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med. 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

[0071] Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer (1989) Proc. Natl. Acad. Sci USA 86:9402-9406; Koes et al. (1995) Proc. Natl. Acad. Sci USA 92:8149-8153; Bensen et al. (1995) Plant Cell 7:75-84). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant gene encoding the instant polypeptides. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous gene encoding the instant polypeptides can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the instant polypeptides disclosed herein.

EXAMPLES

[0072] The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Example 1 Composition of cDNA Libraries; Isolation and Sequencing of cDNA Clones

[0073] cDNA libraries representing mRNAs from various corn, rice, soybean, and wheat tissues were prepared. The characteristics of the libraries are described below. TABLE 2 cDNA Libraries from Corn, Rice, Soybean, and Wheat Library Tissue Clone cbn2 Corn Developing Kernel Two Days After Pollination cbn2.pk0039.c2 p0060 Transgenic Corn Leaf Expressing Gene M1C07 p0060.coran66r (Leucine-Rich Repeat), Family 3-B7, Approximately One Month After Planting in Green House* p0110 Corn (Stages V3/V4**) Leaf Tissue Minus Midrib Harvested p0110.cgsmk69r 4 Hours, 24 Hours and 7 Days After Infiltration With Salicylic Acid, Pooled* r1r24 Rice Leaf 15 Days After Germination, 24 Hours After r1r24.pk0088.f7 Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant r1r6 Rice Leaf 15 Days After Germination, 6 Hours After r1r6.pk0029.d7 Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant r1r6 Rice Leaf 15 Days After Germination, 6 Hours After r1r6.pk0083.f9 Infection of Strain Magaporthe grisea 4360-R-62 (AVR2-YAMO); Resistant rr1 Rice Root of Two Week Old Developing Seedling rr1.pk084.j16 rs11n Rice 15-Day-Old Seedling* rs11n.pk013.h14 rsr9n Rice Leaf 15 Days After Germination Harvested 2-72 Hours rsr9n.pk004.p5 Following Infection With Magnaporta grisea (4360-R-62 and 4360-R-67)* scb1c Soybean Embryogenic Suspension Culture Subjected to 4 scb1c.pk003.d8 Bombardments and Collected 12 Hours Later scb1c Soybean Embryogenic Suspension Culture Subjected to 4 scb1c.pk004.k24 Bombardments and Collected 12 Hours Later ses9c Soybean Embryogenic Suspension ses9c.pk001.j23 sgc2c Soybean Cotyledon 12-20 Days After Germination (Mature sgs2c.pk002.p14 Green) src3c Soybean 8 Day Old Root Infected With Cyst Nematode srr3c.pk003.d10 wdk1c Wheat Developing Kernel, 3 Days After Anthesis wdk1c.pk009.j19 w1e1n Wheat Leaf From 7 Day Old Etiolated Seedling* w1e1n.pk0042.f7 w1k8 Wheat Seedlings 8 Hours After Treatment With Herbicide*** w1k8.pk0006.a4 w1m96 Wheat Seedlings 96 Hours After Inoculation With Erysiphe w1m96.pk032.n8 graminis f. sp tritici wre1n Wheat Root From 7 Day Old Etiolated Seedling* wre1n.pk164.b11

[0074] cDNA libraries may be prepared by any one of many methods available. For example, the cDNAs may be introduced into plasmid vectors by first preparing the cDNA libraries in Uni-ZAP™ XR vectors according to the manufacturer's protocol (Stratagene Cloning Systems, La Jolla, Calif.). The Uni-ZAP™ XR libraries are converted into plasmid libraries according to the protocol provided by Stratagene. Upon conversion, cDNA inserts will be contained in the plasmid vector pBluescript. In addition, the cDNAs may be introduced directly into precut Bluescript II SK(+) vectors (Stratagene) using T4 DNA ligase (New England Biolabs), followed by transfection into DH10B cells according to the manufacturer's protocol (GIBCO BRL Products). Once the cDNA inserts are in plasmid vectors, plasmid DNAs are prepared from randomly picked bacterial colonies containing recombinant pBluescript plasmids, or the insert cDNA sequences are amplified via polymerase chain reaction using primers specific for vector sequences flanking the inserted cDNA sequences. Amplified insert DNAs or plasmid DNAs are sequenced in dye-primer sequencing reactions to generate partial cDNA sequences (expressed sequence tags or “ESTs”; see Adams et al., (1991) Science 252:1651-1656). The resulting ESTs are analyzed using a Perkin Elmer Model 377 fluorescent sequencer.

Example 2 Identification of cDNA Clones

[0075] cDNA clones encoding proteinases were identified by conducting BLAST (Basic Local Alignment Search Tool; Altschul et al. (1993) J. Mol. Biol. 215:403-410; see also www.ncbi.nlm.nih.gov/BLAST/) searches for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences obtained in Example 1 were analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN algorithm provided by the National Center for Biotechnology Information (NCBI). The DNA sequences were translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX algorithm (Gish and States (1993) Nat. Genet. 3:266-272) provided by the NCBI. For convenience, the P-value (probability) of observing a match of a cDNA sequence to a sequence contained in the searched databases merely by chance as calculated by BLAST are reported herein as “pLog” values, which represent the negative of the logarithm of the reported P-value. Accordingly, the greater the pLog value, the greater the likelihood that the cDNA sequence and the BLAST “hit” represent homologous proteins.

Example 3 Characterization of cDNA Clones Encoding Calpain p94

[0076] The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to calpain p94 from Homo sapiens and Drosophila melanogaster (NCBI General Identifier Nos. 1345664 and 600420, respectively). Shown in Table 3 are the BLAST results for individual ESTs (“EST”): TABLE 3 BLAST Results for Sequences Encoding Polypeptides Homologous to Calpain p94 BLAST Clone Status NCBI General Identifier pLog Score cbn2.pk0039.c2 EST 1345664  12.52 rs11n.pk013.h14 EST 600420 11.40 ses9c.pk00l.j23 EST 600420 31.70

[0077] The sequence of the entire cDNA insert in the clones mentioned above was determined. The BLASTP search using the amino acid sequences from clones listed in Table 4 revealed similarity of the polypeptides encoded by the cDNAs to calpain p94 from Drosophila melanogaster (NCBI General Identifier Nos. 600420 and 1079058). Shown in Table 4 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”): TABLE 4 BLAST Results for Sequences Encoding Polypeptides Homologous to Calpain p94 BLAST pLog Score Clone Status 600420 1079058 cbn2.pk0039.c2:fis FIS 43.52 43.52 rs11n.pk013.h14:fis FIS 43.00 43.00 ses9c.pk001.j23:fis FIS 44.05 44.40

[0078] The data in Table 5 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:2, 4, 6, 30, 32, and 34 and the Drosophila melanogaster sequences (NCBI General Identifier Nos. 600420 and 1079058). TABLE 5 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Calpain p94 Percent Identity to SEQ ID NO. 600420 1079058 2 50.0 50.0 4 57.4 57.4 6 52.0 52.0 30 25.8 25.8 32 24.9 24.9 34 26.0 26.0

[0079] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of corn, rice and soybean calpain p94. These sequences represent the first plant sequences encoding calpain p94.

Example 4 Characterization of CDNA Clones Encoding Cysteine Protease 1

[0080] The BLASTX search using the EST sequences from clones listed in Table 3 revealed similarity of the polypeptides encoded by the cDNAs to cysteine protease 1 from Zea mays (NCBI General Identifier No. 1706260). Shown in Table 6 are the BLAST results for individual ESTs (“EST”), or for contigs assembled from two or more ESTs (“Contig”): TABLE 6 BLAST Results for Sequences Encoding Polypeptides Homologous to Cysteine Protease 1 BLAST pLog Score Clone Status 1706260 rr1.pk084.j16 EST 94.52 Contig of: Contig 130.00 wdk1c.pk009.j19 wre1n.pk164.b11

[0081] The entire cDNA insert in clones rrl.pk084.j16 and wdklc.pk009.j19 was determined. The BLASTP search using the amino acid sequences from clones listed in Table 7 revealed similarity of the polypeptides encoded by the cDNAs to cysteine protease 1 from Zea mays (NCBI General Identifier No. 1706260). Shown in Table 7 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”): TABLE 7 BLAST Results for Sequences Encoding Polypeptides Homologous to Cysteine Protease 1 BLAST pLog Score Clone Status 1706260 rr1.pk084.j16:fis FIS 158.00 wdk1c.pk009.j19:fis FIS 110.00

[0082] Amino acid sequence alignments using the Clustal method of alignment indicates that the rice sequence starts 88 amino acids down stream from the corn starting methionine, and that the wheat sequence starts 163 amino acids down stream from the corn starting methionine. The corn sequence has a signal sequence (amino acids 1-19) and a mature protein which corresponds to amino acids 137 through 371. Thus, the rice and wheat sequences included here contain the entire mature protein. The data in Table 8 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:8, 10, 36, and 38 and the Zea mays sequence (NCBI General Identifier No. 1706260). TABLE 8 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Cysteine Protease 1 Percent Identity to SEQ ID NO. 1706260 8 88.0 10 87.1 36 90.9 38 86.3

[0083] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of a rice and a wheat cysteine protease 1. These sequences represent the first rice and wheat sequences encoding cysteine protease 1.

Example 5 Characterization of cDNA Clones Encoding Cysteine Protease 2

[0084] The BLASTX search using the EST sequences from clones listed in Table 9 revealed similarity of the polypeptides encoded by the cDNAs to cysteine protease 2 from Phaseolus vulgaris (NCBI General Identifier No. 2511691). Shown in Table 9 are the BLAST results for sequences of contigs assembled from two or more ESTs (“Contig”): TABLE 9 BLAST Results for Sequences Encoding Polypeptides Homologous to Cysteine Protease 2 BLAST pLog Score Clone Status 2511691 Contig of: Contig 97.70 sgs2c.pk002.p14 srr3c.pk003.d10 scb1c.pk003.d8

[0085] The sequence of the entire cDNA insert in clone srr3c.pk003.d10 was determined. The BLASTP search using the amino acid sequences from clones listed in Table 10 revealed similarity of the polypeptides encoded by the cDNAs to cysteine protease 2 from Phaseolus vulgaris (NCBI General Identifier No.2511691). Shown in Table 10 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones encoding the entire protein (“CGS”): TABLE 10 BLAST Results for Sequences Encoding Polypeptides Homologous to Cysteine Protease 2 BLAST pLog Score Clone Status 2511691 srr3c.pk003.d10:fis CGS 169.00

[0086] The data in Table 11 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:12 and 40 and the Phaseolus vulgaris sequence (NCBI General Identifier No. 2511691). TABLE 11 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to Cysteine Protease 2 Percent Identity to SEQ ID NO. 2511691 12 67.9 40 75.1

[0087] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion and an entire soybean cysteine protease 2. These sequences represent the first soybean sequences encoding cysteine protease 2.

Example 6 Characterization of cDNA Clones Encoding CLP Protease ATP-Binding Subunit

[0088] The BLASTX search using the EST sequences from clones listed in Table 12 revealed similarity of the polypeptides encoded by the cDNAs to two different CLP Protease ATP-binding subunits from Lycopersicon esculentum (NCBI General Identifier Nos. 399213 and 399212). Shown in Table 12 are the BLAST results for individual ESTs (“EST”), or for the sequences of contigs assembled from two or more ESTs (“Contig”): TABLE 12 BLAST Results for Sequences Encoding Polypeptides Homologous to CLP Protease ATP-Binding Subunit BLAST pLog Score Clone Status 399213 399212 p0110.cgsmk69r EST 66.52 66.00 Contig of: Contig 126.00  124.00  rlr6.pk0083.f9 rlr24.pk0088.f7 rlr6.pk0029.d7 wlm96.pk032.n8 EST 98.52 98.70

[0089] The sequence of the entire cDNA insert in clones p0110.cgsmk69r, rlr24.pk0088.f7, and wlm96.pk032.n8 was determined. The BLASTP search using the amino acid sequences from clones listed in Table 13 revealed similarity of the polypeptides encoded by the cDNAs to two different CLP Protease ATP-binding subunits from Lycopersicon esculentum (NCBI General Identifier Nos. 399213 and 399212). Shown in Table 13 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”): TABLE 13 BLAST Results for Sequences Encoding Polypeptides Homologous to CLP Protease ATP-Binding Subunit BLAST pLog Score Clone Status 399213 399212 p0110.cgsmk69r:fis FIS >254.00 >254.00 rlr24.pk0088.f7:fis FIS >254.00 >254.00 wlm96.pk032.n8:fis FIS   140.00   134.00

[0090] The data in Table 14 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:14, 16, 18, 42, 44, and 46 and the Lycopersicon esculentum sequences (NCBI General Identifier Nos. 399213 and 399212). TABLE 14 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to CLP Protease ATP-Binding Subunit Percent Identity to SEQ ID NO. 399213 399212 14 92.2 92.2 16 80.9 79.9 18 86.5 87.1 42 86.9 86.0 44 90.8 89.6 46 88.7 87.4

[0091] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of corn, rice, and soybean CLP protease ATP binding domain. These sequences represent the first corn, rice, and soybean sequences encoding CLP protease ATP binding domain.

Example 7 Characterization of cDNA Clones Encoding CLP Protease Proteolytic Subunit

[0092] The BLASTX search using the EST sequences from clones listed in Table 15 revealed similarity of the polypeptides encoded by the cDNAs to CLPP from Synechococcus PCC7942 (NCBI General Identifier No. 3023500), Myxococcus xanthus (NCBI General Identifier No. 3023519), or Synechocystis sp. (NCBI General Identifier No. 1705930). Shown in Table 15 are the BLAST results for individual ESTs (“EST”): TABLE 15 BLAST Results for Sequences Encoding Polypeptides Homologous to CLP Protease Proteolytic Subunit NCBI General BLAST Clone Status Identifier No. pLog Score p0060.coran66r EST 3023500 36.70 rsr9n.pk004.p5 EST 3023519 43.40 scb1c.pk004.k24 EST 1705930 60.52 wle1n.pk0042.f7 EST 2493737 45.70 wlk8.pk0006.a4 EST 3023519 43.22

[0093] The sequence of the entire cDNA insert in the clones mentioned above was determined. The BLASTP search using the amino acid sequences from clones listed in Table 16 revealed similarity of the polypeptides encoded by the cDNAs to two different CLPP from Arabidopsis thaliana (NCBI General Identifier Nos. 5360593 and 4887543) or from Azospirillum brasilense (NCBI General Identifier No. 6685315). Shown in Table 16 are the BLAST results for the sequences of the entire cDNA inserts comprising the indicated cDNA clones (“FIS”): TABLE 16 BLAST Results for Sequences Encoding Polypeptides Homologous to CLP Protease Proteolytic Subunit NCBI General BLAST Clone Status Identifier No. pLog Score p0060.coran66r:fis FIS 5360593 83.00 rsr9n.pk004.p5:fis FIS 6685315 40.10 scb1c.pk004.k24:fis FIS 4887543 103.00  wle1n.pk0042.f7:fis FIS 4887543 86.00 wlk8.pk0006.a4:fis FIS 6685315 54.05

[0094] The data in Table 17 represents a calculation of the percent identity of the amino acid sequences set forth in SEQ ID NOs:20, 22, 24, 26, 28, 48, 50, 52, 54, and 56 and the Arabidopsis thaliana and Azospirillum brasilense sequences (NCBI General Identifier Nos. 5360593, 4887543, and 6685315). TABLE 17 Percent Identity of Amino Acid Sequences Deduced From the Nucleotide Sequences of cDNA Clones Encoding Polypeptides Homologous to CLP Protease Proteolytic Subunit Percent Identity to SEQ ID NO. 5360593 4887543 6685315 20 78.8 52.2 53.1 22 44.0 55.0 73.4 24 48.7 97.3 58.7 26 55.8 91.6 63.2 28 57.1 64.3 81.0 48 78.8 39.7 38.6 50 34.0 43.8 57.6 52 45.5 92.5 51.5 54 39.0 47.1 60.5 56 48.9 87.9 54.6

[0095] Sequence alignments and percent identity calculations were performed using the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wisc.). Multiple alignment of the sequences was performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method were KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. Sequence alignments and BLAST scores and probabilities indicate that the nucleic acid fragments comprising the instant cDNA clones encode a substantial portion of corn, rice, soybean, and wheat CLPPs. These sequences represent the first corn, rice, soybean, and wheat sequences encoding CLPP.

Example 8 Expression of Chimeric Genes in Monocot Cells

[0096] A chimeric gene comprising a cDNA encoding the instant polypeptides in sense orientation with respect to the maize 27 kD zein promoter that is located 5′ to the cDNA fragment, and the 10 kD zein 3′ end that is located 3′ to the cDNA fragment, can be constructed. The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites (NcoI or SmaI) can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the digested vector pML103 as described below. Amplification is then performed in a standard PCR. The amplified DNA is then digested with restriction enzymes NcoI and SmaI and fractionated on an agarose gel. The appropriate band can be isolated from the gel and combined with a 4.9 kb NcoI-SmaI fragment of the plasmid pML103. Plasmid pML103 has been deposited under the terms of the Budapest Treaty at ATCC (American Type Culture Collection, 10801 University Blvd., Manassas, Va. 20110-2209), and bears accession number ATCC 97366. The DNA segment from pML103 contains a 1.05 kb SalI-NcoI promoter fragment of the maize 27 kD zein gene and a 0.96 kb SmaI-SalI fragment from the 3′ end of the maize 10 kD zein gene in the vector pGem9Zf(+) (Promega). Vector and insert DNA can be ligated at 15° C. overnight, essentially as described (Maniatis). The ligated DNA may then be used to transform E. coli XL1-Blue (Epicurian Coli XL-1 Blue™; Stratagene). Bacterial transformants can be screened by restriction enzyme digestion of plasmid DNA and limited nucleotide sequence analysis using the dideoxy chain termination method (Sequenase™ DNA Sequencing Kit; U.S. Biochemical). The resulting plasmid construct would comprise a chimeric gene encoding, in the 5′ to 3′ direction, the maize 27 kD zein promoter, a cDNA fragment encoding the instant polypeptides, and the 10 kD zein 3′ region.

[0097] The chimeric gene described above can then be introduced into corn cells by the following procedure. Immature corn embryos can be dissected from developing caryopses derived from crosses of the inbred corn lines H99 and LH132. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.

[0098] The plasmid, p35S/Ac (obtained from Dr. Peter Eckes, Hoechst Ag, Frankfurt, Germany) may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0099] The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNAs are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton™ flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic™ PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.

[0100] For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covered a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.

[0101] Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of about 1 cm in diameter of actively growing callus can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.

[0102] Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).

Example 9 Expression of Chimeric Genes in Dicot Cells

[0103] A seed-specific expression cassette composed of the promoter and transcription terminator from the gene encoding the D subunit of the seed storage protein phaseolin from the bean Phaseolus vulgaris (Doyle et al. (1986) J. Biol. Chem. 261:9228-9238) can be used for expression of the instant polypeptides in transformed soybean. The phaseolin cassette includes about 500 nucleotides upstream (5′) from the translation initiation codon and about 1650 nucleotides downstream (3′) from the translation stop codon of phaseolin. Between the 5′ and 3′ regions are the unique restriction endonuclease sites Nco I (which includes the ATG translation initiation codon), Sma I, Kpn I and Xba I. The entire cassette is flanked by Hind III sites.

[0104] The cDNA fragment of this gene may be generated by polymerase chain reaction (PCR) of the cDNA clone using appropriate oligonucleotide primers. Cloning sites can be incorporated into the oligonucleotides to provide proper orientation of the DNA fragment when inserted into the expression vector. Amplification is then performed as described above, and the isolated fragment is inserted into a pUC18 vector carrying the seed expression cassette.

[0105] Soybean embryos may then be transformed with the expression vector comprising sequences encoding the instant polypeptides. To induce somatic embryos, cotyledons, 3-5 mm in length dissected from surface sterilized, immature seeds of the soybean cultivar A2872, can be cultured in the light or dark at 26° C. on an appropriate agar medium for 6-10 weeks. Somatic embryos which produce secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos which multiplied as early, globular staged embryos, the suspensions are maintained as described below.

[0106] Soybean embryogenic suspension cultures can maintained in 35 mL liquid media on a rotary shaker, 150 rpm, at 26° C. with florescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0107] Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic™ PDS 1000/HE instrument (helium retrofit) can be used for these transformations.

[0108] A selectable marker gene which can be used to facilitate soybean transformation is a chimeric gene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al.(1983) Gene 25:179-188) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The seed expression cassette comprising the phaseolin 5′ region, the fragment encoding the instant polypeptides and the phaseolin 3′ region can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.

[0109] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (in order): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂ (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μL 70% ethanol and resuspended in 40 μL of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five μL of the DNA-coated gold particles are then loaded on each macro carrier disk.

[0110] Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.

[0111] Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post bombardment with fresh media containing 50 mg/mL hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.

Example 10 Expression of Chimeric Genes in Microbial Cells

[0112] The cDNAs encoding the instant polypeptides can be inserted into the T7 E. coli expression vector pBT430. This vector is a derivative of pET-3a (Rosenberg et al. (1987) Gene 56:125-135) which employs the bacteriophage T7 RNA polymerase/T7 promoter system. Plasmid pBT430 was constructed by first destroying the EcoR I and Hind III sites in pET-3a at their original positions. An oligonucleotide adaptor containing EcoR I and Hind III sites was inserted at the BamH I site of pET-3a. This created pET-3aM with additional unique cloning sites for insertion of genes into the expression vector. Then, the Nde I site at the position of translation initiation was converted to an Nco I site using oligonucleotide-directed mutagenesis. The DNA sequence of pET-3aM in this region, 5′-CATATGG, was converted to 5′-CCCATGG in pBT430.

[0113] Plasmid DNA containing a cDNA may be appropriately digested to release a nucleic acid fragment encoding the protein. This fragment may then be purified on a 1% NuSieve GTG™ low melting agarose gel (FMC). Buffer and agarose contain 10 μg/ml ethidium bromide for visualization of the DNA fragment. The fragment can then be purified from the agarose gel by digestion with GELase™ (Epicentre Technologies) according to the manufacturer's instructions, ethanol precipitated, dried and resuspended in 20 μL of water. Appropriate oligonucleotide adapters may be ligated to the fragment using T4 DNA ligase (New England Biolabs, Beverly, Mass.). The fragment containing the ligated adapters can be purified from the excess adapters using low melting agarose as described above. The vector pBT430 is digested, dephosphorylated with alkaline phosphatase (NEB) and deproteinized with phenol/chloroform as described above. The prepared vector pBT430 and fragment can then be ligated at 16° C. for 15 hours followed by transformation into DH5 electrocompetent cells (GIBCO BRL). Transformants can be selected on agar plates containing LB media and 100 μg/mL ampicillin. Transformants containing the gene encoding the instant polypeptides are then screened for the correct orientation with respect to the T7 promoter by restriction enzyme analysis.

[0114] For high level expression, a plasmid clone with the cDNA insert in the correct orientation relative to the T7 promoter can be transformed into E. coli strain BL21 (DE3) (Studier et al. (1986) J. Mol. BioL 189:113-130). Cultures are grown in LB medium containing ampicillin (100 mg/L) at 25° C. At an optical density at 600 nm of approximately 1, IPTG (isopropylthio-β-galactoside, the inducer) can be added to a final concentration of 0.4 mM and incubation can be continued for 3 h at 25°. Cells are then harvested by centrifugation and re-suspended in 50 μL of 50 mM Tris-HCl at pH 8.0 containing 0.1 mM DTT and 0.2 mM phenyl methylsulfonyl fluoride. A small amount of 1 mm glass beads can be added and the mixture sonicated 3 times for about 5 seconds each time with a microprobe sonicator. The mixture is centrifuged and the protein concentration of the supernatant determined. One μg of protein from the soluble fraction of the culture can be separated by SDS-polyacrylamide gel electrophoresis. Gels can be observed for protein bands migrating at the expected molecular weight.

[0115] Various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

[0116] The disclosure of each reference set forth above is incorporated herein by reference in its entirety.

1 58 1 304 DNA Zea mays unsure (257) unsure (266) unsure (269) unsure (283) unsure (290) unsure (297) 1 gaaaccagca tttgctacta gtagaaagca aaacgagctt tgggtatcca ttcttgagaa 60 ggcttatgca aaacttcatg gctcttatga ggcattggaa ggtgggcttg ttcaagatgc 120 tctagtcgat ctcacaggag gagctggtga agagattgat atgcgaagtc ctcaagccca 180 acttgatctt gctagtggaa gattgtggtc gcagttgttg catttcaaac aagaagtttt 240 cttcttggtg ctggaantct tcgggntcng aggcccaaat ccnaacaggn gattttnagg 300 gaac 304 2 58 PRT Zea mays 2 Phe Ala Thr Ser Arg Lys Gln Asn Glu Leu Trp Val Ser Ile Leu Glu 1 5 10 15 Lys Ala Tyr Ala Lys Leu His Gly Ser Tyr Glu Ala Leu Glu Gly Gly 20 25 30 Leu Val Gln Asp Ala Leu Val Asp Leu Thr Gly Gly Ala Gly Glu Glu 35 40 45 Ile Asp Met Arg Ser Pro Gln Ala Gln Leu 50 55 3 423 DNA Oryza sativa unsure (128) unsure (198) unsure (204) unsure (250) unsure (263) unsure (293) unsure (311) unsure (319) unsure (328) unsure (346) unsure (365)..(366) unsure (374) unsure (384) unsure (388) unsure (395) unsure (399)..(400) unsure (412)..(413) unsure (422) 3 gggaaaccgg catttgctac tatgtagaaa gcaaaatgag ctgtgggtat ccattcttga 60 aaaagcttat gcaaagcttc atggctctta tgaggcattg gagggtgggc ttgtgcaaga 120 tgctctantg gatctcacag gaggagctgg tgaagagatt gacatgcgga gcccccaagc 180 gcagattgat cttgctantg gaangattgt ggtctcagtt gttgcatttt aaacaagagg 240 gctttcttcn tggggctggg aantccttcc ggcccggatg ctcaatattt cancaagtgg 300 cattgttcaa nggacatgnc cttactcnaa ttttgcaggg taaagnagaa agtttgaatg 360 ggcannaagc ctcngtggca aaanttanaa aaatncccnn gggggccaaa anngaaagtt 420 tnt 423 4 47 PRT Oryza sativa UNSURE (34) 4 Lys Gln Asn Glu Leu Trp Val Ser Ile Leu Glu Lys Ala Tyr Ala Lys 1 5 10 15 Leu His Gly Ser Tyr Glu Ala Leu Glu Gly Gly Leu Val Gln Asp Ala 20 25 30 Leu Xaa Asp Leu Thr Gly Gly Ala Gly Glu Glu Ile Asp Met Arg 35 40 45 5 588 DNA Glycine max unsure (553) unsure (562) unsure (584) 5 gcatttgcta ctagtaagaa gggctatgaa ctttgggtct ccatattgga gaaggcatat 60 gccaagttgc atggctctta tgaagcacta gaaggtgggc ttgttcaaga tgctctggtg 120 gatcttacag ggggtgctgg ggaggaaatt gacatgagga gtggtgaagc ccagattgac 180 cttgcaagtg gtagattgtg gtctcaactg ttgcgcttca agcaagaagg ttttctccta 240 ggtgcaggaa gtccatcagg ttcagatgtg cacatctctt ctagtggcat tgtgcaagga 300 catgcatact caatactgca ggtaagagac gtggatggtc ataaacttgt tcagatccga 360 aatccatggg ccaatgaagt ggagtggaat ggtccctggt ctgactcgtc gcctgagtgg 420 acagatagga taaagcacaa gctgaagcat gttcacagtc aaaagatggc atattctgga 480 tgtcctggca agattttcag attcatttcg ataatatata tttccgttat ctaccatcag 540 agatcgtcat tcngttcatg gncaatggcg ttgttacagt gccngggg 588 6 50 PRT Glycine max 6 Glu Leu Trp Val Ser Ile Leu Glu Lys Ala Tyr Ala Lys Leu His Gly 1 5 10 15 Ser Tyr Glu Ala Leu Glu Gly Gly Leu Val Gln Asp Ala Leu Val Asp 20 25 30 Leu Thr Gly Gly Ala Gly Glu Glu Ile Asp Met Arg Ser Gly Glu Ala 35 40 45 Gln Ile 50 7 1592 DNA Zea mays 7 tttttttttt ttttttttag acagcttcgc atggcaccca aaaaataggt tcattgaatg 60 acatcagatt ccgattacat acagggcaaa ccaatgggaa cagctatggc cgactactcc 120 acgctcaaat cagcaaaact gcagcttgcc aacaagacat ttcacagctg cataagagcc 180 ctatgcgtta acaggaacga caggctacct aacctaactc tatagcattt acagggtaaa 240 cgggacacct atacatagga tgggctacca ggtcagttac cacggctatc tacaaacatt 300 gaaccaagcc tctcaccctg cactttgcgc agctaccaaa gaaccatgga tcatgccttc 360 tctcctttcc cttcagcaca agaagcattg ccgtttgtca gatctgcaac aagcaaccga 420 acttaggtac ttgcatgcag caagattgat tgaagttgtc caggaggcaa ttcctatacc 480 cattatctag ttcccgagaa cccacagctg gatcctgcgt gcacgggtgc ccagcagacg 540 cagctaccat caaacacatg ggatctcaat cttgaactaa acagcctcta gtctgattga 600 tgcttttgaa aaaactgaca aaacaaaagg tgcttcctcc ccagggtgga tggtagttgg 660 cacaattgtg taccctttgg gataaggatc caagaccagt tcgcatgata tctccctcga 720 gttaacgtaa tctgttccac cagcgctttc atgcatgtag atattgtaag cagcacggca 780 gccctgtgtc ttgagtatcc tcattccaat gtaaaacatt gaagaatcat ggctagattg 840 gtagttccga aaaccattcg tctttctaga gaaaccaaca ccctgagtaa gggtaataaa 900 aacgtgaaca gggtatagtg catcacgtcc tgttactcta agtcgatact gtggattttg 960 gtgccacgag tcataatctt ggcaaccacc tgcattgtag ccacgccatt gcccatggac 1020 agagtaacgc atctcaggtg gataaacacg acaaacatat attgaccgaa agtgaatctg 1080 aaaatcttgc caagacatcc agaatacccc attcttcgac tgtggaacat gcatgagctt 1140 atgcttcatc cgttccgtcc actctggtga cgagtctgac catggtccat tccattcaac 1200 ttcatttgcc catggatttc tgatttggat gagtttgtgg ccatcaactt ctcttacctg 1260 caaaattgag tacgcatgtc cctgaacaat gccacttgat gagatgtgag catcagatcc 1320 agaaggactt ccagcaccaa gaagaaaacc ttcttgtttg aaatgcaaca actgcgacca 1380 caatcttcca ctagcaagat caagttgggc ttgaggactt cgcatatcaa tctcttcacc 1440 agctcctcct gtgagatcga ctagagcatc ttgaacaagc ccaccttcca atgcctcata 1500 agagccatga agttttgcat aagccttctc aagaatggat acccaaagct cgttttgctt 1560 tctactagta gcaaatgctg gtttcctcgt gc 1592 8 337 PRT Zea mays 8 Thr Arg Lys Pro Ala Phe Ala Thr Ser Arg Lys Gln Asn Glu Leu Trp 1 5 10 15 Val Ser Ile Leu Glu Lys Ala Tyr Ala Lys Leu His Gly Ser Tyr Glu 20 25 30 Ala Leu Glu Gly Gly Leu Val Gln Asp Ala Leu Val Asp Leu Thr Gly 35 40 45 Gly Ala Gly Glu Glu Ile Asp Met Arg Ser Pro Gln Ala Gln Leu Asp 50 55 60 Leu Ala Ser Gly Arg Leu Trp Ser Gln Leu Leu His Phe Lys Gln Glu 65 70 75 80 Gly Phe Leu Leu Gly Ala Gly Ser Pro Ser Gly Ser Asp Ala His Ile 85 90 95 Ser Ser Ser Gly Ile Val Gln Gly His Ala Tyr Ser Ile Leu Gln Val 100 105 110 Arg Glu Val Asp Gly His Lys Leu Ile Gln Ile Arg Asn Pro Trp Ala 115 120 125 Asn Glu Val Glu Trp Asn Gly Pro Trp Ser Asp Ser Ser Pro Glu Trp 130 135 140 Thr Glu Arg Met Lys His Lys Leu Met His Val Pro Gln Ser Lys Asn 145 150 155 160 Gly Val Phe Trp Met Ser Trp Gln Asp Phe Gln Ile His Phe Arg Ser 165 170 175 Ile Tyr Val Cys Arg Val Tyr Pro Pro Glu Met Arg Tyr Ser Val His 180 185 190 Gly Gln Trp Arg Gly Tyr Asn Ala Gly Gly Cys Gln Asp Tyr Asp Ser 195 200 205 Trp His Gln Asn Pro Gln Tyr Arg Leu Arg Val Thr Gly Arg Asp Ala 210 215 220 Leu Tyr Pro Val His Val Phe Ile Thr Leu Thr Gln Gly Val Gly Phe 225 230 235 240 Ser Arg Lys Thr Asn Gly Phe Arg Asn Tyr Gln Ser Ser His Asp Ser 245 250 255 Ser Met Phe Tyr Ile Gly Met Arg Ile Leu Lys Thr Gln Gly Cys Arg 260 265 270 Ala Ala Tyr Asn Ile Tyr Met His Glu Ser Ala Gly Gly Thr Asp Tyr 275 280 285 Val Asn Ser Arg Glu Ile Ser Cys Glu Leu Val Leu Asp Pro Tyr Pro 290 295 300 Lys Gly Tyr Thr Ile Val Pro Thr Thr Ile His Pro Gly Glu Glu Ala 305 310 315 320 Pro Phe Val Leu Ser Val Phe Ser Lys Ala Ser Ile Arg Leu Glu Ala 325 330 335 Val 9 1670 DNA Oryza sativa 9 gcacgagggg aaaccggcat ttgctactag tagaaagcaa aatgagctgt gggtatccat 60 tcttgaaaaa gcttatgcaa agcttcatgg ctcttatgag gcattggagg gtgggcttgt 120 gcaagatgct ctagtggatc tcacaggagg agctggtgaa gagattgaca tgcggagccc 180 ccaagcgcag attgatcttg ctagtggaag attgtggtct cagttgttgc attttaaaca 240 agagggcttt cttcttggtg ctggaagtcc ttctggctcg gatgctcata tttcatcaag 300 tggcattgtt cagggacatg cttactcaat tttgcaggta agagaagttg atggccataa 360 gctcgtgcaa attagaaatc cgtgggcaaa tgaagttgaa tggaatggtc cgtggtcaga 420 ctcatcacaa gagtggactg agcgaatgaa gcacaaactt aagcatgttc cacagtcaaa 480 gaatggggta ttctggatgt cttggcaaga ttttcagatc cactttcggt caatatatgt 540 ctgtcgtgtt tatccacccg agatgcgtta ctctgtccat ggccaatggc gtggttatag 600 tgcaggtggt tgccaagatt atgactcatg gcatcaaaat cctcagtacc ggcttagagt 660 aacaggacgg gatgcactgt atcctgtaca tgtatttatt acccttacgc agggtgttgg 720 tttttctaga aagacaaatg gtttccggaa ctatcaatct agccatgact cttctatgtt 780 ttacattgga atgaggatac tcaagacacg gggctgccgt gctgcttaca atatctacat 840 gcatgaatca gtgggtggaa cagattatgt taactcaagg gagatatcat gtgaattagt 900 gttggagcct tatccaaaag gatacacaat tgtgcccaca accatccatc ctggagagga 960 agcacctttt gttttatcag tttttacaaa agcaccaatc aagctagaag ctgtttaatg 1020 caagattcat actagatgtg ttcgctgctg gtagctgtgc cttgctgttg ctggcccagg 1080 ctcctttggg gttcaagtga tgcagattcc agctgtgggt tcaagaggat cagataatgg 1140 tgtttgctgc agcaatatgc cggtacctgg gagatatgaa gcagcagctc tgctcttgaa 1200 cctgaacatt ctgggcttca tcagacgcta catgcaaccg cataagcttg attgcttcct 1260 aacgtgacag gaaaggcaat ctttctgtca gcaggcatgg tgttgataca tcagaatcat 1320 ttgatcgccg tgcatgactc tggatgtttt ctagtcgatg cttgtagata gtggtggcaa 1380 ctaacattta gacctggtag ccccatcccg tgtataggta tcccatgtca ccgtgtacat 1440 actatagaat tagatgagtt aaccttgtgg tgtcgtttcc tgttagcgaa atagagctct 1500 tgtgcagctg ggaaatacca ttttagcatg ctgcagtttt gctgatcaag gcgagcagtc 1560 gtcggccata gcgttttcat tgattgcctg tatgtaatcg aaatctgatc tcattcaatg 1620 gaagctattt tttggtaccc tttgaagcaa aaaaaaaaaa aaaaaaaaaa 1670 10 338 PRT Oryza sativa 10 His Glu Gly Lys Pro Ala Phe Ala Thr Ser Arg Lys Gln Asn Glu Leu 1 5 10 15 Trp Val Ser Ile Leu Glu Lys Ala Tyr Ala Lys Leu His Gly Ser Tyr 20 25 30 Glu Ala Leu Glu Gly Gly Leu Val Gln Asp Ala Leu Val Asp Leu Thr 35 40 45 Gly Gly Ala Gly Glu Glu Ile Asp Met Arg Ser Pro Gln Ala Gln Ile 50 55 60 Asp Leu Ala Ser Gly Arg Leu Trp Ser Gln Leu Leu His Phe Lys Gln 65 70 75 80 Glu Gly Phe Leu Leu Gly Ala Gly Ser Pro Ser Gly Ser Asp Ala His 85 90 95 Ile Ser Ser Ser Gly Ile Val Gln Gly His Ala Tyr Ser Ile Leu Gln 100 105 110 Val Arg Glu Val Asp Gly His Lys Leu Val Gln Ile Arg Asn Pro Trp 115 120 125 Ala Asn Glu Val Glu Trp Asn Gly Pro Trp Ser Asp Ser Ser Gln Glu 130 135 140 Trp Thr Glu Arg Met Lys His Lys Leu Lys His Val Pro Gln Ser Lys 145 150 155 160 Asn Gly Val Phe Trp Met Ser Trp Gln Asp Phe Gln Ile His Phe Arg 165 170 175 Ser Ile Tyr Val Cys Arg Val Tyr Pro Pro Glu Met Arg Tyr Ser Val 180 185 190 His Gly Gln Trp Arg Gly Tyr Ser Ala Gly Gly Cys Gln Asp Tyr Asp 195 200 205 Ser Trp His Gln Asn Pro Gln Tyr Arg Leu Arg Val Thr Gly Arg Asp 210 215 220 Ala Leu Tyr Pro Val His Val Phe Ile Thr Leu Thr Gln Gly Val Gly 225 230 235 240 Phe Ser Arg Lys Thr Asn Gly Phe Arg Asn Tyr Gln Ser Ser His Asp 245 250 255 Ser Ser Met Phe Tyr Ile Gly Met Arg Ile Leu Lys Thr Arg Gly Cys 260 265 270 Arg Ala Ala Tyr Asn Ile Tyr Met His Glu Ser Val Gly Gly Thr Asp 275 280 285 Tyr Val Asn Ser Arg Glu Ile Ser Cys Glu Leu Val Leu Glu Pro Tyr 290 295 300 Pro Lys Gly Tyr Thr Ile Val Pro Thr Thr Ile His Pro Gly Glu Glu 305 310 315 320 Ala Pro Phe Val Leu Ser Val Phe Thr Lys Ala Pro Ile Lys Leu Glu 325 330 335 Ala Val 11 1550 DNA Glycine max 11 gcacgaggca tttgctacta gtaagaaggg ctatgaactt tgggtctcca tattggagaa 60 ggcatatgcc aagttgcatg gctcttatga agcactagaa ggtgggcttg ttcaagatgc 120 tctggtggat cttacagggg gtgctgggga ggaaattgac atgaggagtg gtgaagccca 180 gattgacctt gcaagtggta gattgtggtc tcaactgttg cgcttcaagc aagaaggttt 240 tctcctaggt gcaggaagtc catcaggttc agatgtgcac atctcttcta gtggcattgt 300 gcaaggacat gcatactcaa tactgcaggt aagagacgtg gatggtcata aacttgttca 360 gatccgaaat ccatgggcca atgaagtgga gtggaatggt ccctggtctg actcgtcgcc 420 tgagtggaca gataggataa agcacaagct gaagcatgtt ccacagtcaa aagatggcat 480 attctggatg tcctggcaag attttcagat tcattttcga tcaatatata tttgccgtat 540 ctacccatca gagatgcgtc attctgttca tggtcaatgg cgtggttaca gtgccggggg 600 gtgtcaggat tatgatacgt ggaatcaaaa tccacagttc agattgactt caactgggca 660 agatgcatca tttccaattc atgtattcat taccttaact cagggtgtgg gattttcaag 720 aacaacagct ggttttagaa attatcaatc cagccatgat tcacagatgt tttacattgg 780 aatgaggata ctaaaaactc gtggcagacg tgctgctttc aatatatacc tacatgaatc 840 agttggtggg acagactatg ttaattcacg agaaatatcc tgtgaaatgg ttttggaacc 900 tgagccaaag ggatatacta tagttcctac tactatacac cccggtgaag aagcaccgtt 960 tgtactttct gttttcacca aggcgtcgat aactctggaa gctttgtagt gcctagggat 1020 agtttttaca tgtatcttgt cctcttgata agtttctctg cctgggttct cggtggatca 1080 tttacttgta ctgccggagc ccgtctttag aacaatcggc attgagatac tattcctggc 1140 ggaaacgaca tggcatctgt ttgagagatg aatgaggtat agctgcgcat aaactcttgg 1200 tctttgaatc tgacgatttg tcatcttgaa caatgcttct gccagcattg aagagggctc 1260 tcggggtgtt tattgtgtac ataaaaaatt ggtactatag gggtatactt gtaaccattt 1320 aagcaaagtt gaaaaagaaa tagctgaaaa taagtaggaa attactaaca cctggttcaa 1380 tggaggtaag gacggtgtgg ggaggtatag taacaagcat tgagtgactg attgtaaatt 1440 cagttgccgt tttgacaact gcaaaaaatt gtacaaacat taacaattat cagttcctat 1500 caaaaaaaaa aaaaaataac tcgagggggg gccgtaccaa atctttcccg 1550 12 335 PRT Glycine max 12 His Glu Ala Phe Ala Thr Ser Lys Lys Gly Tyr Glu Leu Trp Val Ser 1 5 10 15 Ile Leu Glu Lys Ala Tyr Ala Lys Leu His Gly Ser Tyr Glu Ala Leu 20 25 30 Glu Gly Gly Leu Val Gln Asp Ala Leu Val Asp Leu Thr Gly Gly Ala 35 40 45 Gly Glu Glu Ile Asp Met Arg Ser Gly Glu Ala Gln Ile Asp Leu Ala 50 55 60 Ser Gly Arg Leu Trp Ser Gln Leu Leu Arg Phe Lys Gln Glu Gly Phe 65 70 75 80 Leu Leu Gly Ala Gly Ser Pro Ser Gly Ser Asp Val His Ile Ser Ser 85 90 95 Ser Gly Ile Val Gln Gly His Ala Tyr Ser Ile Leu Gln Val Arg Asp 100 105 110 Val Asp Gly His Lys Leu Val Gln Ile Arg Asn Pro Trp Ala Asn Glu 115 120 125 Val Glu Trp Asn Gly Pro Trp Ser Asp Ser Ser Pro Glu Trp Thr Asp 130 135 140 Arg Ile Lys His Lys Leu Lys His Val Pro Gln Ser Lys Asp Gly Ile 145 150 155 160 Phe Trp Met Ser Trp Gln Asp Phe Gln Ile His Phe Arg Ser Ile Tyr 165 170 175 Ile Cys Arg Ile Tyr Pro Ser Glu Met Arg His Ser Val His Gly Gln 180 185 190 Trp Arg Gly Tyr Ser Ala Gly Gly Cys Gln Asp Tyr Asp Thr Trp Asn 195 200 205 Gln Asn Pro Gln Phe Arg Leu Thr Ser Thr Gly Gln Asp Ala Ser Phe 210 215 220 Pro Ile His Val Phe Ile Thr Leu Thr Gln Gly Val Gly Phe Ser Arg 225 230 235 240 Thr Thr Ala Gly Phe Arg Asn Tyr Gln Ser Ser His Asp Ser Gln Met 245 250 255 Phe Tyr Ile Gly Met Arg Ile Leu Lys Thr Arg Gly Arg Arg Ala Ala 260 265 270 Phe Asn Ile Tyr Leu His Glu Ser Val Gly Gly Thr Asp Tyr Val Asn 275 280 285 Ser Arg Glu Ile Ser Cys Glu Met Val Leu Glu Pro Glu Pro Lys Gly 290 295 300 Tyr Thr Ile Val Pro Thr Thr Ile His Pro Gly Glu Glu Ala Pro Phe 305 310 315 320 Val Leu Ser Val Phe Thr Lys Ala Ser Ile Thr Leu Glu Ala Leu 325 330 335 13 505 DNA Oryza sativa unsure (120) unsure (283) unsure (299) unsure (337) unsure (479) 13 ccgcggagca cggcgtcacc aagttctccg acctcacccc ggccgagttc cgccgggcct 60 acctcggcct ccgcacgtcg cgccgcgcct tcctgcgggg gctcggcggg tccgcccacn 120 aggcgcccgt cctccccacc gacggcctcc ccgacgactt cgactggaga gaccacggcg 180 ccgtcggccc cgtcaagaac cagggatcgt gcgggtcgtg ctggtcgttc agcgcgtcgg 240 gggcgctaga gggagcgaac tacctggcga cgggcaagat ggncgtgctc tccgagcanc 300 agatggtcga ttgcgaccat gagtgtgatt catcatnaac ctgattcatg tgatgctgga 360 tgcaatggtg gattgatgac taacgccttc agctatcttt tgaaatccgg tggccttgag 420 agtgagaagg attaccccta cactgggagg gatggcacct gcaaatttga caagtcgang 480 attgttactt cagttcaaaa cttca 505 14 167 PRT Oryza sativa UNSURE (40) UNSURE (94) UNSURE (99) UNSURE (112) UNSURE (159) 14 Ala Glu His Gly Val Thr Lys Phe Ser Asp Leu Thr Pro Ala Glu Phe 1 5 10 15 Arg Arg Ala Tyr Leu Gly Leu Arg Thr Ser Arg Arg Ala Phe Leu Arg 20 25 30 Gly Leu Gly Gly Ser Ala His Xaa Ala Pro Val Leu Pro Thr Asp Gly 35 40 45 Leu Pro Asp Asp Phe Asp Trp Arg Asp His Gly Ala Val Gly Pro Val 50 55 60 Lys Asn Gln Gly Ser Cys Gly Ser Cys Trp Ser Phe Ser Ala Ser Gly 65 70 75 80 Ala Leu Glu Gly Ala Asn Tyr Leu Ala Thr Gly Lys Met Xaa Val Leu 85 90 95 Ser Glu Xaa Gln Met Val Asp Cys Asp His Glu Cys Asp Ser Ser Xaa 100 105 110 Pro Asp Ser Cys Asp Ala Gly Cys Asn Gly Gly Leu Met Thr Asn Ala 115 120 125 Phe Ser Tyr Leu Leu Lys Ser Gly Gly Leu Glu Ser Glu Lys Asp Tyr 130 135 140 Pro Tyr Thr Gly Arg Asp Gly Thr Cys Lys Phe Asp Lys Ser Xaa Ile 145 150 155 160 Val Thr Ser Val Gln Asn Phe 165 15 717 DNA Triticum aestivum unsure (342) unsure (634) 15 gtcgttcagc gcgtccgggg cgttggaggg agccaactac ctggccacgg gcaagatgga 60 ggtgctctcc gagcagcagc tggtcgactg cgaccatgag tgcgacccag cagaacctga 120 ttcatgcgat gctggatgca atggtgggtt gatgacttca gcctttagct atctgttgaa 180 atctggtggc cttgagagag aaaaggatta cccttacacc gggaaggacg gtacctgcaa 240 atttgagaag tccaagattg ctgcttcagt tcaaaacttc agcgttgtcg ctgttgatga 300 agaacagatt gctgctaacc ttgtgaaata tggaccgctg gncatcggta tcaacgccgc 360 atacatgcag acatacatcg gcggagtgtc atgcccatac atctgcggca ggcacctcga 420 ccacggtgtc cttctcgtcg gctacggggc gtctggcttc gcgccttccc gcttcaagga 480 gaagccctac tggatcatca agaactcatg gggcgagaac tggggggaca agggttacta 540 caagatctgc aggggctcga acgtgcgcaa caagtgtggc gtcgactcca tggtctccac 600 ggtgtccgcc actcacgcct ccaaggacga gtangctctg ggtctgatct gatctgatcg 660 gcgggcctcc tggtgtcatc ttgggttccg tgtgtgtatc gctagaaaga aacttta 717 16 209 PRT Triticum aestivum UNSURE (114) 16 Ser Phe Ser Ala Ser Gly Ala Leu Glu Gly Ala Asn Tyr Leu Ala Thr 1 5 10 15 Gly Lys Met Glu Val Leu Ser Glu Gln Gln Leu Val Asp Cys Asp His 20 25 30 Glu Cys Asp Pro Ala Glu Pro Asp Ser Cys Asp Ala Gly Cys Asn Gly 35 40 45 Gly Leu Met Thr Ser Ala Phe Ser Tyr Leu Leu Lys Ser Gly Gly Leu 50 55 60 Glu Arg Glu Lys Asp Tyr Pro Tyr Thr Gly Lys Asp Gly Thr Cys Lys 65 70 75 80 Phe Glu Lys Ser Lys Ile Ala Ala Ser Val Gln Asn Phe Ser Val Val 85 90 95 Ala Val Asp Glu Glu Gln Ile Ala Ala Asn Leu Val Lys Tyr Gly Pro 100 105 110 Leu Xaa Ile Gly Ile Asn Ala Ala Tyr Met Gln Thr Tyr Ile Gly Gly 115 120 125 Val Ser Cys Pro Tyr Ile Cys Gly Arg His Leu Asp His Gly Val Leu 130 135 140 Leu Val Gly Tyr Gly Ala Ser Gly Phe Ala Pro Ser Arg Phe Lys Glu 145 150 155 160 Lys Pro Tyr Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Asp 165 170 175 Lys Gly Tyr Tyr Lys Ile Cys Arg Gly Ser Asn Val Arg Asn Lys Cys 180 185 190 Gly Val Asp Ser Met Val Ser Thr Val Ser Ala Thr His Ala Ser Lys 195 200 205 Asp 17 1174 DNA Oryza sativa 17 gcacgagccg cggagcacgg cgtcaccaag ttctccgacc tcaccccggc cgagttccgc 60 cgggcctacc tcggcctccg cacgtcgcgc cgcgccttcc tgcgggggct cggcgggtcc 120 gcccacgagg cgcccgtcct ccccaccgac ggcctccccg acgacttcga ctggagagac 180 cacggcgccg tcggccccgt caagaaccag ggatcgtgcg ggtcgtgctg gtcgttcagc 240 gcgtcggggg cgctagaggg agcgaactac ctggcgacgg gcaagatgga cgtgctctcc 300 gagcagcaga tggtcgattg cgaccatgag tgtgattcat cagaacctga ttcatgtgat 360 gctggatgca atggtggatt gatgactaac gccttcagct atcttttgaa atccggtggc 420 cttgagagtg agaaggatta cccctacact gggagggatg gcacctgcaa atttgacaag 480 tcgaagattg ttacttcagt tcagaacttc agtgttgtct ctgtcgatga ggatcagatt 540 gctgccaacc ttgtcaaaca tgggccactt gcaattggca tcaatgctgc gtacatgcaa 600 acatacattg gtggtgtttc gtgcccgtac atctgtggca ggcaccttga tcacggtgtt 660 cttctcgttg gctacggcgc atctggtttt gctccaatcc gcctaaagga taaggcctac 720 tggatcatca agaactcctg gggcgagaac tggggagagc atgggtacta caagatctgc 780 aggggctcca acgtccgcaa caaatgtggc gtggattcta tggtctccac cgtgtctgcc 840 atccacacct caaaggagta gattctgatc agtagtcccc cgaccatcct gtggatggtt 900 cacagttggt gattctgata ttatatataa gctagaacta cgaaatatac ttagtttatg 960 ctccatctgc gctgttattg cagttatgat aagcagcgat gatgtgaagc tgcaactgaa 1020 tgtttgtcct aagttatatg cttggtttgc tacgcaatgc tacacgctat ttggaggtag 1080 ctttaagtat tatcgccatt cacgaacttg tatttttact attaccaatc ttttgaatgg 1140 tctgtattat atgcaaaaaa aaaaaaaaaa aaaa 1174 18 286 PRT Oryza sativa 18 Ala Arg Ala Ala Glu His Gly Val Thr Lys Phe Ser Asp Leu Thr Pro 1 5 10 15 Ala Glu Phe Arg Arg Ala Tyr Leu Gly Leu Arg Thr Ser Arg Arg Ala 20 25 30 Phe Leu Arg Gly Leu Gly Gly Ser Ala His Glu Ala Pro Val Leu Pro 35 40 45 Thr Asp Gly Leu Pro Asp Asp Phe Asp Trp Arg Asp His Gly Ala Val 50 55 60 Gly Pro Val Lys Asn Gln Gly Ser Cys Gly Ser Cys Trp Ser Phe Ser 65 70 75 80 Ala Ser Gly Ala Leu Glu Gly Ala Asn Tyr Leu Ala Thr Gly Lys Met 85 90 95 Asp Val Leu Ser Glu Gln Gln Met Val Asp Cys Asp His Glu Cys Asp 100 105 110 Ser Ser Glu Pro Asp Ser Cys Asp Ala Gly Cys Asn Gly Gly Leu Met 115 120 125 Thr Asn Ala Phe Ser Tyr Leu Leu Lys Ser Gly Gly Leu Glu Ser Glu 130 135 140 Lys Asp Tyr Pro Tyr Thr Gly Arg Asp Gly Thr Cys Lys Phe Asp Lys 145 150 155 160 Ser Lys Ile Val Thr Ser Val Gln Asn Phe Ser Val Val Ser Val Asp 165 170 175 Glu Asp Gln Ile Ala Ala Asn Leu Val Lys His Gly Pro Leu Ala Ile 180 185 190 Gly Ile Asn Ala Ala Tyr Met Gln Thr Tyr Ile Gly Gly Val Ser Cys 195 200 205 Pro Tyr Ile Cys Gly Arg His Leu Asp His Gly Val Leu Leu Val Gly 210 215 220 Tyr Gly Ala Ser Gly Phe Ala Pro Ile Arg Leu Lys Asp Lys Ala Tyr 225 230 235 240 Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp Gly Glu His Gly Tyr 245 250 255 Tyr Lys Ile Cys Arg Gly Ser Asn Val Arg Asn Lys Cys Gly Val Asp 260 265 270 Ser Met Val Ser Thr Val Ser Ala Ile His Thr Ser Lys Glu 275 280 285 19 935 DNA Triticum aestivum 19 gcacgaggtc gttcagcgcg tccggggcgt tggagggagc caactacctg gccacgggca 60 agatggaggt gctctccgag cagcagctgg tcgactgcga ccatgagtgc gacccagcag 120 aacctgattc atgcgatgct ggatgcaatg gtgggttgat gacttcagcc tttagctatc 180 tgttgaaatc tggtggcctt gagagagaaa aggattaccc ttacaccggg aaggacggta 240 cctgcaaatt tgagaagtcc aagattgctg cttcagttca aaacttcagc gttgtcgctg 300 ttgatgaaga acagattgct gctaaccttg tgaaatatgg accgctggcc atcggtatca 360 acgccgcata catgcagaca tacatcggcg gagtgtcatg cccatacatc tgcggcaggc 420 acctcgacca cggtgtcctt ctcgtcggct acggggcgtc tggcttcgcg ccttcccgct 480 tcaaggagaa gccctactgg atcatcaaga actcatgggg cgagaactgg ggggacaagg 540 gttactacaa gatctgcagg ggctcgaacg tgcgcaacaa gtgtggcgtc gactccatgg 600 tctccacggt gtccgccact cacgcctcca aggacgagta ggctctggtc tgatctgatc 660 tgatcggcgg ccctcctggt gtcgatcttg gtttcggtgt gtgtatcgct agaaagaaac 720 tttaatacgt agtagtcggc taggctccat cgtcgttgtg gtatcagcag cgaagatgcg 780 aagtcgcaat agaatgcttg ctgtataact tatgcatttg ctaaatttgc tacgccatgc 840 atgtctgcca cacgctattt ggatgtggct aaagaactcc tgaataattc tgtacataat 900 ttgtattgct tccatcaaaa aaaaaaaaaa aaaaa 935 20 212 PRT Triticum aestivum 20 Thr Arg Ser Phe Ser Ala Ser Gly Ala Leu Glu Gly Ala Asn Tyr Leu 1 5 10 15 Ala Thr Gly Lys Met Glu Val Leu Ser Glu Gln Gln Leu Val Asp Cys 20 25 30 Asp His Glu Cys Asp Pro Ala Glu Pro Asp Ser Cys Asp Ala Gly Cys 35 40 45 Asn Gly Gly Leu Met Thr Ser Ala Phe Ser Tyr Leu Leu Lys Ser Gly 50 55 60 Gly Leu Glu Arg Glu Lys Asp Tyr Pro Tyr Thr Gly Lys Asp Gly Thr 65 70 75 80 Cys Lys Phe Glu Lys Ser Lys Ile Ala Ala Ser Val Gln Asn Phe Ser 85 90 95 Val Val Ala Val Asp Glu Glu Gln Ile Ala Ala Asn Leu Val Lys Tyr 100 105 110 Gly Pro Leu Ala Ile Gly Ile Asn Ala Ala Tyr Met Gln Thr Tyr Ile 115 120 125 Gly Gly Val Ser Cys Pro Tyr Ile Cys Gly Arg His Leu Asp His Gly 130 135 140 Val Leu Leu Val Gly Tyr Gly Ala Ser Gly Phe Ala Pro Ser Arg Phe 145 150 155 160 Lys Glu Lys Pro Tyr Trp Ile Ile Lys Asn Ser Trp Gly Glu Asn Trp 165 170 175 Gly Asp Lys Gly Tyr Tyr Lys Ile Cys Arg Gly Ser Asn Val Arg Asn 180 185 190 Lys Cys Gly Val Asp Ser Met Val Ser Thr Val Ser Ala Thr His Ala 195 200 205 Ser Lys Asp Glu 210 21 743 DNA Glycine max unsure (645) unsure (680) unsure (740) 21 tgcacctttc tcttcctccg atggctaatc tctcactctt gttcttcggt ctcctcctat 60 tctccgctgc cgtagccacc gtcgaacgaa tcgacgatga agacaacctt ctgatccgtc 120 aagtggtgcc ggacgcggag gaccaccacc tgctcaacgc ggagcaccac ttctccgcct 180 tcaagacaaa gttcgccaag acctacgcca cgcaggagga gcacgaccac cgcttccgta 240 tcttcaagaa caacttgctc cgcgccaagt cgcaccagaa attggacccc tccgccgtcc 300 acggcgtcac caggttctcc gatctcactc cggctgagtt tcgcggccag ttcctcggcc 360 tgaagccgct ccgccttccc tccgacgctc agaaggctcc gatccttccg accagcgacc 420 ttcctaccga tttcgattgg cgcgaccatg gagctgttac cggcgtcaag aatcagggct 480 cgtgcggatc gtgttggtca tttagcgccg ttggagcttt ggaaggtgcc cattttcttt 540 ctaccggtgg gctcgtgagc ctcagtgagc agcaacttgt ggattgcgat catgagtgtg 600 atccggaaga acgtggagca tgtgattcgg gttgtaacgg tgggntgatg accactgcat 660 tttgagtaca cactcaaggn tggtggacta atgccaagaa agaggattat ccctacaatg 720 ggagaaaacg ttggccctgn aaa 743 22 234 PRT Glycine max UNSURE (209) UNSURE (220) 22 Met Ala Asn Leu Ser Leu Leu Phe Phe Gly Leu Leu Leu Phe Ser Ala 1 5 10 15 Ala Val Ala Thr Val Glu Arg Ile Asp Asp Glu Asp Asn Leu Leu Ile 20 25 30 Arg Gln Val Val Pro Asp Ala Glu Asp His His Leu Leu Asn Ala Glu 35 40 45 His His Phe Ser Ala Phe Lys Thr Lys Phe Ala Lys Thr Tyr Ala Thr 50 55 60 Gln Glu Glu His Asp His Arg Phe Arg Ile Phe Lys Asn Asn Leu Leu 65 70 75 80 Arg Ala Lys Ser His Gln Lys Leu Asp Pro Ser Ala Val His Gly Val 85 90 95 Thr Arg Phe Ser Asp Leu Thr Pro Ala Glu Phe Arg Gly Gln Phe Leu 100 105 110 Gly Leu Lys Pro Leu Arg Leu Pro Ser Asp Ala Gln Lys Ala Pro Ile 115 120 125 Leu Pro Thr Ser Asp Leu Pro Thr Asp Phe Asp Trp Arg Asp His Gly 130 135 140 Ala Val Thr Gly Val Lys Asn Gln Gly Ser Cys Gly Ser Cys Trp Ser 145 150 155 160 Phe Ser Ala Val Gly Ala Leu Glu Gly Ala His Phe Leu Ser Thr Gly 165 170 175 Gly Leu Val Ser Leu Ser Glu Gln Gln Leu Val Asp Cys Asp His Glu 180 185 190 Cys Asp Pro Glu Glu Arg Gly Ala Cys Asp Ser Gly Cys Asn Gly Gly 195 200 205 Xaa Met Thr Thr Ala Phe Glu Tyr Thr Leu Lys Xaa Gly Gly Leu Met 210 215 220 Lys Lys Glu Asp Tyr Pro Tyr Asn Gly Arg 225 230 23 1369 DNA Glycine max 23 cggcacgagt gcacctttct cttcctccga tggctaatct ctcactcttg ttcttcggtc 60 tcctcctatt ctccgctgcc gtagccaccg tcgaacgaat cgacgatgaa gacaaccttc 120 tgatccgtca agtggtgccg gacgcggagg accaccacct gctcaacgcg gagcaccact 180 tctccgcctt caagacaaag ttcgccaaga cctacgccac gcaggaggag cacgaccacc 240 gcttccgtat cttcaagaac aacttgctcc gcgccaagtc gcaccagaaa ttggacccct 300 ccgccgtcca cggcgtcacc aggttctccg atctcactcc gtctgagttt cgcggccagt 360 tcctcggcct gaagccgctc cgccttccct ccgacgctca gaaggctccg atccttccga 420 ccagcgacct tcctaccgat ttcgattggc gcgaccatgg agctgttacc ggcgtcaaga 480 atcagggctc gtgcggatgg tgttggtcat ttagcgccgt tggagctttg gaaggtgccc 540 attttctttc taccggtggg ctcgtgagcc tcagtgagca gcaacttgtg gattgcgatc 600 atgagtgtga tccggaagag cgtggagcat gtgattcggg ttgtaacggt gggttgatga 660 ccactgcatt tgagtacaca ctcaaggctg gtggactaat gcgagaagag gattatccct 720 acactggaag agaccgtggc ccctgcaaat ttgacaagag caaaatcgct gcttccgtgg 780 ctaatttcag tgtggtttcc cttgatgaag aacaaattgc tgcaaatctg gtcaagaatg 840 gtcctcttgc agttggtatc aatgcagttt ttatgcagac atatattggt ggcgtctcat 900 gcccatacat ctgcggcaag catttggatc atggggttct tttggtgggc tatggatctg 960 gtgcttatgc tccaattcgt tttaaggaaa agccttactg gatcataaag aattcatggg 1020 gggagagctg gggagaagaa ggatattaca agatctgcag aggtcgcaat gtatgtgggg 1080 tggactcgat ggtctcaact gtagctgcta tacatgtttc taaccattaa atataaggat 1140 ggatgcctaa acatggtagg ggcaccagta tagtgtgtat gtaaataatt tacatgatgt 1200 ataatgttat ggaggaggaa actgctaagc ccatgtttat gcttttatgc tgtaattctc 1260 tatgctagct agtctagcta caaatattac ccacggttat cgatagttat tgcaagtaac 1320 ctgaataaaa ttaatttgtg ttcccacaat taaaaaaaaa aaaaaaaaa 1369 24 366 PRT Glycine max 24 Met Ala Asn Leu Ser Leu Leu Phe Phe Gly Leu Leu Leu Phe Ser Ala 1 5 10 15 Ala Val Ala Thr Val Glu Arg Ile Asp Asp Glu Asp Asn Leu Leu Ile 20 25 30 Arg Gln Val Val Pro Asp Ala Glu Asp His His Leu Leu Asn Ala Glu 35 40 45 His His Phe Ser Ala Phe Lys Thr Lys Phe Ala Lys Thr Tyr Ala Thr 50 55 60 Gln Glu Glu His Asp His Arg Phe Arg Ile Phe Lys Asn Asn Leu Leu 65 70 75 80 Arg Ala Lys Ser His Gln Lys Leu Asp Pro Ser Ala Val His Gly Val 85 90 95 Thr Arg Phe Ser Asp Leu Thr Pro Ser Glu Phe Arg Gly Gln Phe Leu 100 105 110 Gly Leu Lys Pro Leu Arg Leu Pro Ser Asp Ala Gln Lys Ala Pro Ile 115 120 125 Leu Pro Thr Ser Asp Leu Pro Thr Asp Phe Asp Trp Arg Asp His Gly 130 135 140 Ala Val Thr Gly Val Lys Asn Gln Gly Ser Cys Gly Trp Cys Trp Ser 145 150 155 160 Phe Ser Ala Val Gly Ala Leu Glu Gly Ala His Phe Leu Ser Thr Gly 165 170 175 Gly Leu Val Ser Leu Ser Glu Gln Gln Leu Val Asp Cys Asp His Glu 180 185 190 Cys Asp Pro Glu Glu Arg Gly Ala Cys Asp Ser Gly Cys Asn Gly Gly 195 200 205 Leu Met Thr Thr Ala Phe Glu Tyr Thr Leu Lys Ala Gly Gly Leu Met 210 215 220 Arg Glu Glu Asp Tyr Pro Tyr Thr Gly Arg Asp Arg Gly Pro Cys Lys 225 230 235 240 Phe Asp Lys Ser Lys Ile Ala Ala Ser Val Ala Asn Phe Ser Val Val 245 250 255 Ser Leu Asp Glu Glu Gln Ile Ala Ala Asn Leu Val Lys Asn Gly Pro 260 265 270 Leu Ala Val Gly Ile Asn Ala Val Phe Met Gln Thr Tyr Ile Gly Gly 275 280 285 Val Ser Cys Pro Tyr Ile Cys Gly Lys His Leu Asp His Gly Val Leu 290 295 300 Leu Val Gly Tyr Gly Ser Gly Ala Tyr Ala Pro Ile Arg Phe Lys Glu 305 310 315 320 Lys Pro Tyr Trp Ile Ile Lys Asn Ser Trp Gly Glu Ser Trp Gly Glu 325 330 335 Glu Gly Tyr Tyr Lys Ile Cys Arg Gly Arg Asn Val Cys Gly Val Asp 340 345 350 Ser Met Val Ser Thr Val Ala Ala Ile His Val Ser Asn His 355 360 365 25 441 DNA Zea mays unsure (362) unsure (375) unsure (398) 25 gccaagaaca atttctgctt gattggagag cctggtgttg gaaaaactgc aattgctgaa 60 ggacttgctc agcgcatttc tacaggcgat gtacctgaaa caatagaagg gaaaaaggtc 120 ataacccttg acatgggact tcttgttgct ggcacaaagt accgtggaga attcgaagaa 180 agattaaaga agctgatgga ggaaataaag caaagtgatg agataatact ctttattgat 240 gaagttcaca ctctgatagg agcaggagca gcggaggtgc tatagatgct gctaatatct 300 tgaagcctgc gttgccagag gtgaattaca gtgcattgga gccactacac tagatgaata 360 tnggaagccc attgngaaag acccgccttg acggaggntt caacctgtga aagtgccaga 420 ccaacagtag atgaaaccat t 441 26 128 PRT Zea mays UNSURE (121) UNSURE (125) 26 Lys Asn Asn Phe Cys Leu Ile Gly Glu Pro Gly Val Gly Lys Thr Ala 1 5 10 15 Ile Ala Glu Gly Leu Ala Gln Arg Ile Ser Thr Gly Asp Val Pro Glu 20 25 30 Thr Ile Glu Gly Lys Lys Val Ile Thr Leu Asp Met Gly Leu Leu Val 35 40 45 Ala Gly Thr Lys Tyr Arg Gly Glu Phe Glu Glu Arg Leu Lys Lys Leu 50 55 60 Met Glu Glu Ile Lys Gln Ser Asp Glu Ile Ile Leu Phe Ile Asp Glu 65 70 75 80 Val His Thr Leu Ile Gly Ala Gly Ala Ala Glu Gly Ala Ile Asp Ala 85 90 95 Ala Asn Ile Leu Glu Ala Cys Val Ala Arg Gly Glu Leu Gln Cys Ile 100 105 110 Gly Ala Thr Thr Leu Asp Glu Tyr Xaa Lys Pro Ile Xaa Lys Asp Pro 115 120 125 27 2471 DNA Oryza sativa 27 tttcgttgct gtcgaaatac cattcacacc acgtgcaaaa cgtgttttgg agctttcatt 60 ggaagaagct cgtcagctag gacacaacta tattggatct gagcacttgc ttcttggact 120 gctccgtgag ggtgaaggtg tagcagcccg tgtgctcgaa agccttggag ccgatcctag 180 caatattcgc acgcaggtta tccgaatgat tggcgagact acagaagctg ttggtgcagg 240 agttggagga gggagtagtg gcaataaaat gccaacactt gaggagtacg gaactaattt 300 aacaaaatta gcagaggagg gaaagctaga tcctgttgtt ggaaggcaac cccagattga 360 gcgtgtcgta caaattctgg gcagacgaac aaagaacaac ccatgcttaa ttggagagcc 420 tggtgttgga aagacagcaa ttgcagaagg ccttgctcaa cgcatttcta ctggtgatgt 480 gcctgaaaca attgaaggaa agaaggtcat tacccttgat atgggacttc ttgttgctgg 540 tacaaaatac cgtggagaat ttgaagaaag attaaagaag ctgatggaag aaatcaagca 600 gagtgatgag ataatactat ttattgatga agtccacact ctcataggag caggagcagc 660 tgagggtgct attgacgctg ctaacatttt aaagccagca ttagcaagag gagaactaca 720 gtgtattgga gccaccacac ttgatgaata caggaagcat attgagaaag acccagcatt 780 agaaagacgt ttccagcctg taagagtgcc agagccaaca gttgatgaaa ccatagaaat 840 tctcagaggg cttcgggaac gatatgagat ccatcataaa cttcgttaca ctgatgatgc 900 tctgatttca gctgccaagc tatcttatca atacatcagt gatcgtttcc tcccagataa 960 agcaattgat ttgattgatg aagcaggttc acgtgtaagg cttcgacatg cccaggttcc 1020 tgaagaagct agagagcttg acaaggagct caagcaaatc acaaaagata agaatgaagc 1080 tgtccgtagc caggacttcg aaaaggctgg agagttacgt gatcgtgaaa tggaattgaa 1140 ggcccagata acagctctca ttgacaagag caaggagatg agcaaagcag agactgaatc 1200 aggggagaca gggccactgg tcaatgaagc agatatccag cacattgtat cctcgtggac 1260 tggtattcca gtagagaagg tatcaagtga cgagtccgat aagcttctta agatggaaga 1320 gactttgcat cagcgtgtca ttggtcaaga tgaggctgtg aaagccataa gtcgctccat 1380 ccgccgtgct cgtgtgggcc tcaagaaccc gaacaggccg attgcaagct tcattttcgc 1440 aggtccaacc ggtgttggta aatccgagct cgcaaaagca cttgcagcat attactttgg 1500 atctgaggag gccatgatca ggcttgatat gagtgaattc atggagaggc acactgtatc 1560 caagttgatt ggttcacccc cagggtatgt tgggtacacg gagggtggac agctgactga 1620 ggcagttcga cgcaggccat acacagtcgt gcttttcgac gagatcgaaa aggcgcatcc 1680 agatgtattc aacatgatgc tccagatctt ggaagatgga aggctgactg acagcaaggg 1740 aagaacagtg gacttcaaga acacacttct cataatgact tcgaacgtcg gaagcagcgt 1800 catcgagaag ggtggtcgga agataggttt cgatctcgat tacgatgaga aggacagcag 1860 ctacagcagg atcaagagcc ttgtcgtcga ggagatgaag cagtacttcc gccccgagtt 1920 cctcaaccgt ctcgacgaga tgatcgtctt caggcaactc accaagctgg aggtcaagga 1980 gatcgccgag atcatgctca aggaggtctt tgacaggctc aaggccaagg acattgacct 2040 ccaggtcacc gagaagttca aggagcgtat cgttgacgaa ggcttcaacc cgagctatgg 2100 tgcgaggccg ctaaggaggg ccatcatgag gctcctggag gacagcctcg cggagaagat 2160 gctagctggg gaggtgaagg agggtgattc tgccattgtc gatgtggatt ccgaggggaa 2220 ggtgattgta ctgaatggcc aaagtgggtt gcctgagctt tcaactccgg ctgtcactgt 2280 gtagtagttc atatatactg cagagtgtta agagatgcag tgcttttcat tcagatatat 2340 ttctgcatag ttagcaactt agcataactg tatatatagt atatacaaat caaaggagga 2400 ggaaacacca gctgattcct ggttaaaaaa aaaagaaaaa aaaaaaaaaa aaaaaaaaaa 2460 aaaaaaaaaa a 2471 28 760 PRT Oryza sativa 28 Phe Val Ala Val Glu Ile Pro Phe Thr Pro Arg Ala Lys Arg Val Leu 1 5 10 15 Glu Leu Ser Leu Glu Glu Ala Arg Gln Leu Gly His Asn Tyr Ile Gly 20 25 30 Ser Glu His Leu Leu Leu Gly Leu Leu Arg Glu Gly Glu Gly Val Ala 35 40 45 Ala Arg Val Leu Glu Ser Leu Gly Ala Asp Pro Ser Asn Ile Arg Thr 50 55 60 Gln Val Ile Arg Met Ile Gly Glu Thr Thr Glu Ala Val Gly Ala Gly 65 70 75 80 Val Gly Gly Gly Ser Ser Gly Asn Lys Met Pro Thr Leu Glu Glu Tyr 85 90 95 Gly Thr Asn Leu Thr Lys Leu Ala Glu Glu Gly Lys Leu Asp Pro Val 100 105 110 Val Gly Arg Gln Pro Gln Ile Glu Arg Val Val Gln Ile Leu Gly Arg 115 120 125 Arg Thr Lys Asn Asn Pro Cys Leu Ile Gly Glu Pro Gly Val Gly Lys 130 135 140 Thr Ala Ile Ala Glu Gly Leu Ala Gln Arg Ile Ser Thr Gly Asp Val 145 150 155 160 Pro Glu Thr Ile Glu Gly Lys Lys Val Ile Thr Leu Asp Met Gly Leu 165 170 175 Leu Val Ala Gly Thr Lys Tyr Arg Gly Glu Phe Glu Glu Arg Leu Lys 180 185 190 Lys Leu Met Glu Glu Ile Lys Gln Ser Asp Glu Ile Ile Leu Phe Ile 195 200 205 Asp Glu Val His Thr Leu Ile Gly Ala Gly Ala Ala Glu Gly Ala Ile 210 215 220 Asp Ala Ala Asn Ile Leu Lys Pro Ala Leu Ala Arg Gly Glu Leu Gln 225 230 235 240 Cys Ile Gly Ala Thr Thr Leu Asp Glu Tyr Arg Lys His Ile Glu Lys 245 250 255 Asp Pro Ala Leu Glu Arg Arg Phe Gln Pro Val Arg Val Pro Glu Pro 260 265 270 Thr Val Asp Glu Thr Ile Glu Ile Leu Arg Gly Leu Arg Glu Arg Tyr 275 280 285 Glu Ile His His Lys Leu Arg Tyr Thr Asp Asp Ala Leu Ile Ser Ala 290 295 300 Ala Lys Leu Ser Tyr Gln Tyr Ile Ser Asp Arg Phe Leu Pro Asp Lys 305 310 315 320 Ala Ile Asp Leu Ile Asp Glu Ala Gly Ser Arg Val Arg Leu Arg His 325 330 335 Ala Gln Val Pro Glu Glu Ala Arg Glu Leu Asp Lys Glu Leu Lys Gln 340 345 350 Ile Thr Lys Asp Lys Asn Glu Ala Val Arg Ser Gln Asp Phe Glu Lys 355 360 365 Ala Gly Glu Leu Arg Asp Arg Glu Met Glu Leu Lys Ala Gln Ile Thr 370 375 380 Ala Leu Ile Asp Lys Ser Lys Glu Met Ser Lys Ala Glu Thr Glu Ser 385 390 395 400 Gly Glu Thr Gly Pro Leu Val Asn Glu Ala Asp Ile Gln His Ile Val 405 410 415 Ser Ser Trp Thr Gly Ile Pro Val Glu Lys Val Ser Ser Asp Glu Ser 420 425 430 Asp Lys Leu Leu Lys Met Glu Glu Thr Leu His Gln Arg Val Ile Gly 435 440 445 Gln Asp Glu Ala Val Lys Ala Ile Ser Arg Ser Ile Arg Arg Ala Arg 450 455 460 Val Gly Leu Lys Asn Pro Asn Arg Pro Ile Ala Ser Phe Ile Phe Ala 465 470 475 480 Gly Pro Thr Gly Val Gly Lys Ser Glu Leu Ala Lys Ala Leu Ala Ala 485 490 495 Tyr Tyr Phe Gly Ser Glu Glu Ala Met Ile Arg Leu Asp Met Ser Glu 500 505 510 Phe Met Glu Arg His Thr Val Ser Lys Leu Ile Gly Ser Pro Pro Gly 515 520 525 Tyr Val Gly Tyr Thr Glu Gly Gly Gln Leu Thr Glu Ala Val Arg Arg 530 535 540 Arg Pro Tyr Thr Val Val Leu Phe Asp Glu Ile Glu Lys Ala His Pro 545 550 555 560 Asp Val Phe Asn Met Met Leu Gln Ile Leu Glu Asp Gly Arg Leu Thr 565 570 575 Asp Ser Lys Gly Arg Thr Val Asp Phe Lys Asn Thr Leu Leu Ile Met 580 585 590 Thr Ser Asn Val Gly Ser Ser Val Ile Glu Lys Gly Gly Arg Lys Ile 595 600 605 Gly Phe Asp Leu Asp Tyr Asp Glu Lys Asp Ser Ser Tyr Ser Arg Ile 610 615 620 Lys Ser Leu Val Val Glu Glu Met Lys Gln Tyr Phe Arg Pro Glu Phe 625 630 635 640 Leu Asn Arg Leu Asp Glu Met Ile Val Phe Arg Gln Leu Thr Lys Leu 645 650 655 Glu Val Lys Glu Ile Ala Glu Ile Met Leu Lys Glu Val Phe Asp Arg 660 665 670 Leu Lys Ala Lys Asp Ile Asp Leu Gln Val Thr Glu Lys Phe Lys Glu 675 680 685 Arg Ile Val Asp Glu Gly Phe Asn Pro Ser Tyr Gly Ala Arg Pro Leu 690 695 700 Arg Arg Ala Ile Met Arg Leu Leu Glu Asp Ser Leu Ala Glu Lys Met 705 710 715 720 Leu Ala Gly Glu Val Lys Glu Gly Asp Ser Ala Ile Val Asp Val Asp 725 730 735 Ser Glu Gly Lys Val Ile Val Leu Asn Gly Gln Ser Gly Leu Pro Glu 740 745 750 Leu Ser Thr Pro Ala Val Thr Val 755 760 29 540 DNA Triticum aestivum unsure (434) unsure (462) unsure (495) unsure (515) 29 cttcttcttc tcaatcacgc tgctcccaac atttgatgtc attatcagga gcgtgttctt 60 gaagtccact gttctcccct tgctgtcggt taaccttccg tcttccagga tctggagcat 120 catgttgaac acatccggat gtgccttctc aatctcatca aaaagcacaa cgctgtatgg 180 ccgccgtcga accgcctccg tcagctgccc accttcagtg tatcccacat agcctggtgg 240 tgaaccgatc aacttggaca cagtgtgcct ctccatgaac tcactcatat ccagccggat 300 catggcttct tcagagccga agtaatatga tgccagagtc tttgcaagct ctgatttccc 360 aacaccagtg ggacctgcaa aaatgaagct cgcaattggt ctgttggggc tcttgagggc 420 cacacgagca cggngaacag accgacttat tgctttcaca gnctcgtctt gggcgatgac 480 acgcttatgc aatgnctcct tcaacctaaa gaagnttatc aaattcgcag tcgagacttt 540 30 178 PRT Triticum aestivum UNSURE (9) UNSURE (16) UNSURE (27) UNSURE (36) 30 Lys Val Ser Thr Ala Asn Leu Ile Xaa Phe Phe Arg Leu Lys Glu Xaa 1 5 10 15 Leu His Lys Arg Val Ile Ala Gln Asp Glu Xaa Val Lys Ala Ile Ser 20 25 30 Arg Ser Val Xaa Arg Ala Arg Val Ala Leu Lys Ser Pro Asn Arg Pro 35 40 45 Ile Ala Ser Phe Ile Phe Ala Gly Pro Thr Gly Val Gly Lys Ser Glu 50 55 60 Leu Ala Lys Thr Leu Ala Ser Tyr Tyr Phe Gly Ser Glu Glu Ala Met 65 70 75 80 Ile Arg Leu Asp Met Ser Glu Phe Met Glu Arg His Thr Val Ser Lys 85 90 95 Leu Ile Gly Ser Pro Pro Gly Tyr Val Gly Tyr Thr Glu Gly Gly Gln 100 105 110 Leu Thr Glu Ala Val Arg Arg Arg Pro Tyr Ser Val Val Leu Phe Asp 115 120 125 Glu Ile Glu Lys Ala His Pro Asp Val Phe Asn Met Met Leu Gln Ile 130 135 140 Leu Glu Asp Gly Arg Leu Thr Asp Ser Lys Gly Arg Thr Val Asp Phe 145 150 155 160 Lys Asn Thr Leu Leu Ile Met Thr Ser Asn Val Gly Ser Ser Val Ile 165 170 175 Glu Lys 31 2050 DNA Zea mays 31 ccacgcgtcc gccaagaaca atccctgctt gattggagag cctggtgttg gaaaaactgc 60 aattgctgaa ggacttgctc agcgcatttc tacaggcgat gtacctgaaa caatagaagg 120 gaaaaaggtc ataacccttg acatgggact tcttgttgct ggcacaaagt accgtggaga 180 attcgaagaa agattaaaga agctgatgga ggaaataaag caaagtgatg agataatact 240 ctttattgat gaagttcaca ctctgatagg agcaggagca gcggaggtgc tatagatgct 300 gctaatatct tgaagcctgc gttggccaga ggtgaattac agtgcattgg agccactaca 360 ctagatgaat ataggaagca cattgagaaa gacccagcac ttgaacggag gtttcaacct 420 gtgaaagtgc cagaaccaac agtagatgaa accattgaaa tcctcagagg actgagggaa 480 cgatatgaga tccaccataa acttcgttac actgatgaag ctctgattgc agctgcaaag 540 ctgtcatatc aatatatcag tgatcggttt ctcccagata aggcaattga cttgattgat 600 gaagcaggtt cccgtgttag gctacagcat gcacaggtcc ccgaggaagc aagagagctt 660 gacaaggagc tcaaacaagt cacgaaacag aagaatgaag ctgttcgaag ccaggatttt 720 gagaaggctg gggaattgag agaccgtgaa atggaattga aggcccagat aacagccctc 780 attgacaaga gcaaggaatt gagcaaagca gaggaagagt ctggagagac aggacctatg 840 gtcaatgaag aagatatcca gcacatagta tcttcatgga ctggcatccc tgtggagaag 900 gtttccagcg atgaatctga taagcttctt aagatggaag agactttgca caagcgtgtc 960 attggccaag atgaggctgt ggtagcaatt agtcgctcca tccgccgtgc tcgtgtgggt 1020 ctcaagaacc ccaacaggcc aattgcaagc tttatttttg ctggtcccac cggcgttggg 1080 aagtctgagc ttgcaaaggc tcttgcagcc tattactttg gctctgagga ggctatgatc 1140 cggcttgata tgagtgaatt catggagaga cacacggtat ccaagctgat tggttcacct 1200 ccaggatatg taggatacac tgagggtggc cagctgacag aggcagttcg acggcggcca 1260 tacacagttg tgctctttga tgagattgag aaggcacacc ctgatgtctt caacatgatg 1320 cttcagattt tggaagatgg gagattgact gacagcaagg gaaggacggt ggacttcaag 1380 aacacactcc tgatcatgac ctcaaatgta gggagcagtg tcatcgagaa gggtggaagg 1440 aagatcggat ttgaccttga ctctgatgag aaggacagta gctacagcag gatcaagagc 1500 ctggtcatcg aggagatgaa gcagtatttc cgacctgagt tcctcaaccg tctcgatgag 1560 atgatcgtgt tcaggcagct taccaagctc gaggtcaagg agatagcgga catcatgctc 1620 caggaggtct ttgacaggct gaaggccaag gacatcaatc ttcaagtgac cgagaagttc 1680 aaggagcggg tggtggacga aggctacaac cctagctatg gtgcacgccc gctgaggcga 1740 gccatcatga ggctgctgga ggacagcctt gctgagaaga tgctcgcagg ggaggtaaag 1800 gagggcgact ctgccatagt agatgtggac tcggagggga aggttgttgt gctcaatggt 1860 cagggcggca taccggagct ctcaactccg gcgatcaccg tttagctcgt acataacaaa 1920 tgacaaaatt aatagcatag tttttgttca aacacattat catttatggt tagaatatct 1980 gtgtatatgt agtggtatag tcaatgggga aatcgttcct gcctctaaaa aaaaaaaaaa 2040 aaaaaaaaag 2050 32 550 PRT Zea mays 32 Ser Ser His Ser Asp Arg Ser Arg Ser Ser Gly Gly Ala Ile Asp Ala 1 5 10 15 Ala Asn Ile Leu Lys Pro Ala Leu Ala Arg Gly Glu Leu Gln Cys Ile 20 25 30 Gly Ala Thr Thr Leu Asp Glu Tyr Arg Lys His Ile Glu Lys Asp Pro 35 40 45 Ala Leu Glu Arg Arg Phe Gln Pro Val Lys Val Pro Glu Pro Thr Val 50 55 60 Asp Glu Thr Ile Glu Ile Leu Arg Gly Leu Arg Glu Arg Tyr Glu Ile 65 70 75 80 His His Lys Leu Arg Tyr Thr Asp Glu Ala Leu Ile Ala Ala Ala Lys 85 90 95 Leu Ser Tyr Gln Tyr Ile Ser Asp Arg Phe Leu Pro Asp Lys Ala Ile 100 105 110 Asp Leu Ile Asp Glu Ala Gly Ser Arg Val Arg Leu Gln His Ala Gln 115 120 125 Val Pro Glu Glu Ala Arg Glu Leu Asp Lys Glu Leu Lys Gln Val Thr 130 135 140 Lys Gln Lys Asn Glu Ala Val Arg Ser Gln Asp Phe Glu Lys Ala Gly 145 150 155 160 Glu Leu Arg Asp Arg Glu Met Glu Leu Lys Ala Gln Ile Thr Ala Leu 165 170 175 Ile Asp Lys Ser Lys Glu Leu Ser Lys Ala Glu Glu Glu Ser Gly Glu 180 185 190 Thr Gly Pro Met Val Asn Glu Glu Asp Ile Gln His Ile Val Ser Ser 195 200 205 Trp Thr Gly Ile Pro Val Glu Lys Val Ser Ser Asp Glu Ser Asp Lys 210 215 220 Leu Leu Lys Met Glu Glu Thr Leu His Lys Arg Val Ile Gly Gln Asp 225 230 235 240 Glu Ala Val Val Ala Ile Ser Arg Ser Ile Arg Arg Ala Arg Val Gly 245 250 255 Leu Lys Asn Pro Asn Arg Pro Ile Ala Ser Phe Ile Phe Ala Gly Pro 260 265 270 Thr Gly Val Gly Lys Ser Glu Leu Ala Lys Ala Leu Ala Ala Tyr Tyr 275 280 285 Phe Gly Ser Glu Glu Ala Met Ile Arg Leu Asp Met Ser Glu Phe Met 290 295 300 Glu Arg His Thr Val Ser Lys Leu Ile Gly Ser Pro Pro Gly Tyr Val 305 310 315 320 Gly Tyr Thr Glu Gly Gly Gln Leu Thr Glu Ala Val Arg Arg Arg Pro 325 330 335 Tyr Thr Val Val Leu Phe Asp Glu Ile Glu Lys Ala His Pro Asp Val 340 345 350 Phe Asn Met Met Leu Gln Ile Leu Glu Asp Gly Arg Leu Thr Asp Ser 355 360 365 Lys Gly Arg Thr Val Asp Phe Lys Asn Thr Leu Leu Ile Met Thr Ser 370 375 380 Asn Val Gly Ser Ser Val Ile Glu Lys Gly Gly Arg Lys Ile Gly Phe 385 390 395 400 Asp Leu Asp Ser Asp Glu Lys Asp Ser Ser Tyr Ser Arg Ile Lys Ser 405 410 415 Leu Val Ile Glu Glu Met Lys Gln Tyr Phe Arg Pro Glu Phe Leu Asn 420 425 430 Arg Leu Asp Glu Met Ile Val Phe Arg Gln Leu Thr Lys Leu Glu Val 435 440 445 Lys Glu Ile Ala Asp Ile Met Leu Gln Glu Val Phe Asp Arg Leu Lys 450 455 460 Ala Lys Asp Ile Asn Leu Gln Val Thr Glu Lys Phe Lys Glu Arg Val 465 470 475 480 Val Asp Glu Gly Tyr Asn Pro Ser Tyr Gly Ala Arg Pro Leu Arg Arg 485 490 495 Ala Ile Met Arg Leu Leu Glu Asp Ser Leu Ala Glu Lys Met Leu Ala 500 505 510 Gly Glu Val Lys Glu Gly Asp Ser Ala Ile Val Asp Val Asp Ser Glu 515 520 525 Gly Lys Val Val Val Leu Asn Gly Gln Gly Gly Ile Pro Glu Leu Ser 530 535 540 Thr Pro Ala Ile Thr Val 545 550 33 740 DNA Oryza sativa unsure (628) unsure (674) unsure (740) 33 tttcgttgct gtcgaaatac cattcacacc acgtgcaaaa cgtgttttgg agctttcatt 60 ggaagaagct cgtcagctag gacacaacta tattggatct gagcacttgc ttcttggact 120 gctccgtgag ggtgaaggtg tagcagcccg tgtgctcgaa agccttggag ccgatcctag 180 caatattcgc acgcaggtta tccgaatgat tggcgagact acagaagctg ttggtgcagg 240 agttggagga gggagtagtg gcaataaaat gccaacactt gaggagtacg gaactaattt 300 aacaaaatta gcagaggagg gaaagctaga tcctgttgtt ggaaggcaac cccagattga 360 gcgtgtcgta caaattctgg ggcagacgaa caaagaacaa cccatgcctt aattggagaa 420 cctggtgttt ggaaaagaca gcaattgcag aaggccttgc tcaacgcatt tctactggtg 480 atgtgcctga aacaattgaa ggaaagaagg tcattaccct tgatatggga cttcttgttg 540 ctggtacaaa ataccgtgga gaatttgaag aaagattaaa gaagctgatg gaagaaatca 600 agcagagtga tgagataata ctatttantg atgaagtcca cactctcata ggagcaggag 660 caactgaggg tgcnattgac gctgctaaca ttttaagcca cattacaaga ggagaactac 720 atgttttgga gccacacacn 740 34 298 PRT Oryza sativa UNSURE (65)..(66)..(67)..(68) UNSURE (276) 34 Phe Thr Pro Arg Ala Lys Arg Val Leu Glu Leu Ser Leu Glu Glu Ala 1 5 10 15 Arg Gln Leu Gly His Asn Tyr Ile Gly Ser Glu His Leu Leu Leu Gly 20 25 30 Leu Leu Arg Glu Gly Glu Gly Val Ala Ala Arg Val Leu Glu Ser Leu 35 40 45 Gly Ala Asp Pro Ser Asn Ile Arg Thr Gln Val Ile Arg Met Ile Gly 50 55 60 Xaa Xaa Xaa Xaa Phe Val Ala Val Glu Ile Pro Phe Thr Pro Arg Ala 65 70 75 80 Lys Arg Val Leu Glu Leu Ser Leu Glu Glu Ala Arg Gln Leu Gly His 85 90 95 Asn Tyr Ile Gly Ser Glu His Leu Leu Leu Gly Leu Leu Arg Glu Gly 100 105 110 Glu Gly Val Ala Ala Arg Val Leu Glu Ser Leu Gly Ala Asp Pro Ser 115 120 125 Asn Ile Arg Thr Gln Val Ile Arg Met Ile Gly Glu Thr Thr Glu Ala 130 135 140 Val Gly Ala Gly Val Gly Gly Gly Ser Ser Gly Asn Lys Met Pro Thr 145 150 155 160 Leu Glu Glu Tyr Gly Thr Asn Leu Thr Lys Leu Ala Glu Glu Gly Lys 165 170 175 Leu Asp Pro Val Val Gly Arg Gln Pro Arg Leu Ser Val Ser Tyr Lys 180 185 190 Phe Trp Gly Arg Arg Thr Lys Asn Asn Pro Cys Leu Ile Gly Glu Pro 195 200 205 Gly Val Trp Lys Thr Ala Ile Ala Glu Gly Leu Ala Gln Arg Ile Ser 210 215 220 Thr Gly Asp Val Pro Glu Thr Ile Glu Gly Lys Lys Val Ile Thr Leu 225 230 235 240 Asp Met Gly Leu Leu Val Ala Gly Thr Lys Tyr Arg Gly Glu Phe Glu 245 250 255 Glu Arg Leu Lys Lys Leu Met Glu Glu Ile Lys Gln Ser Asp Glu Ile 260 265 270 Ile Leu Phe Xaa Asp Glu Val His Thr Leu Ile Gly Ala Gly Ala Thr 275 280 285 Glu Gly Ala Ile Asp Ala Ala Asn Ile Leu 290 295 35 1205 DNA Triticum aestivum 35 ctcgtgccga attcggcacg aggtggacta ctatattttg aattctctta atgctgatag 60 agcaacccaa ctgtttaaaa acttcatgtg ggatgttaat ccaccatatt taacttgttt 120 agagtgttca ttgatataat tggaagatga catgtaattt catagtatga tctaggcgtt 180 cttgtcggtg cggtcggtct cagttgatga taaaaaatgt ttgtcatact tctgacatta 240 aatagttatc actgcaagta aattattact agtgtccttg aacctgcctt ttctctagca 300 taaaaaccgc actagtgtat gtttattcta ttcatgtggg ttgatgatct caactttctg 360 gatgccaacc accatatatc tgcactttct ttgatataga tgctaactaa tagttgctat 420 taatatattc cctttatcga aaaaaaacta atggttgctg tgcctgttgc aatgttatgc 480 cattaggctg gagagttgcg agatcgtgaa atggaattga aggcgccaga taacagcctt 540 gattgacaag agcaaggaga tgaacaaagc agagactgag tcgggagaga cggggccgat 600 ggtgcatgaa tcagatatcc agcacattgt gtcatcatgg actggtattc cagtggagaa 660 agtctcgact gacgaatctg ataaacttct taagatggaa gagacattgc ataagcgtgt 720 catcggccaa gacgaggctg tgaaagcaat aagtcggtct gttcgccgtg ctcgtgtggg 780 cctcaagagc cccaacagac caattgcgag cttcattttt gcaggtccca ctggtgttgg 840 gaaatcagag cttgcaaaga ctctggcatc atattacttc ggctctgaag aagccatgat 900 ccggctggat atgagtgagt tcatggagag gcacactgtg tccaagttga tcggttcacc 960 accaggctat gtgggataca ctgaaggtgg gcagctgacg gaggcggttc gacggcggcc 1020 atacagcgtt gtgctttttg atgagattga gaaggcacat ccggatgtgt tcaacatgat 1080 gctccagatc ctggaagacg gaaggttaac cgacagcaag gggagaacag tggacttcaa 1140 gaacacgctc ctgataatga catcaaatgt tgggagcagc gtgattgaga agaagaagct 1200 cgtgc 1205 36 239 PRT Triticum aestivum 36 Ala Gly Glu Leu Arg Asp Arg Glu Met Glu Leu Arg Arg Gln Ile Thr 1 5 10 15 Ala Leu Ile Asp Lys Ser Lys Glu Met Asn Lys Ala Glu Thr Glu Ser 20 25 30 Gly Glu Thr Gly Pro Met Val His Glu Ser Asp Ile Gln His Ile Val 35 40 45 Ser Ser Trp Thr Gly Ile Pro Val Glu Lys Val Ser Thr Asp Glu Ser 50 55 60 Asp Lys Leu Leu Lys Met Glu Glu Thr Leu His Lys Arg Val Ile Gly 65 70 75 80 Gln Asp Glu Ala Val Lys Ala Ile Ser Arg Ser Val Arg Arg Ala Arg 85 90 95 Val Gly Leu Lys Ser Pro Asn Arg Pro Ile Ala Ser Phe Ile Phe Ala 100 105 110 Gly Pro Thr Gly Val Gly Lys Ser Glu Leu Ala Lys Thr Leu Ala Ser 115 120 125 Tyr Tyr Phe Gly Ser Glu Glu Ala Met Ile Arg Leu Asp Met Ser Glu 130 135 140 Phe Met Glu Arg His Thr Val Ser Lys Leu Ile Gly Ser Pro Pro Gly 145 150 155 160 Tyr Val Gly Tyr Thr Glu Gly Gly Gln Leu Thr Glu Ala Val Arg Arg 165 170 175 Arg Pro Tyr Ser Val Val Leu Phe Asp Glu Ile Glu Lys Ala His Pro 180 185 190 Asp Val Phe Asn Met Met Leu Gln Ile Leu Glu Asp Gly Arg Leu Thr 195 200 205 Asp Ser Lys Gly Arg Thr Val Asp Phe Lys Asn Thr Leu Leu Ile Met 210 215 220 Thr Ser Asn Val Gly Ser Ser Val Ile Glu Lys Lys Lys Leu Val 225 230 235 37 498 DNA Zea mays unsure (327) unsure (350) unsure (359) unsure (372) unsure (397) unsure (423) unsure (448) unsure (459) unsure (486) unsure (492) 37 agctcctcct ccttgacgcc atcgacccgg actctgacat ccgcctcttc gtcaactcac 60 cagggggatc ccttagcgca acaatggcca tctatgatgt aatgcagctt gtgagggcag 120 acgtgtccac tattggaatg ggcatagctg gatcaacagc ttctataatc cttggtggtg 180 gcacgaaggg caagcgattt gccatgccca acaccaggat tatgatccat cagcctgtcg 240 gaggtgcaag cgggcaggcc ctagatgtag aggtccaagc gaaggagata ttgaccaaca 300 agaggaatgt tcatcggatc gtatcangct tcacaggccg cactcctgan ccagtagana 360 aagacttgac anagatcgta caggggcctc tcgaggngtc gataggatca tgatgctgat 420 cgntgagaat atatccattg agctgtcnga gaggtgaanc taatacatag aagacgtaca 480 gtcacnagtt cntacaca 498 38 113 PRT Zea mays UNSURE (109) 38 Leu Leu Leu Leu Asp Ala Ile Asp Pro Asp Ser Asp Ile Arg Leu Phe 1 5 10 15 Val Asn Ser Pro Gly Gly Ser Leu Ser Ala Thr Met Ala Ile Tyr Asp 20 25 30 Val Met Gln Leu Val Arg Ala Asp Val Ser Thr Ile Gly Met Gly Ile 35 40 45 Ala Gly Ser Thr Ala Ser Ile Ile Leu Gly Gly Gly Thr Lys Gly Lys 50 55 60 Arg Phe Ala Met Pro Asn Thr Arg Ile Met Ile His Gln Pro Val Gly 65 70 75 80 Gly Ala Ser Gly Gln Ala Leu Asp Val Glu Val Gln Ala Lys Glu Ile 85 90 95 Leu Thr Asn Lys Arg Asn Val His Arg Ile Val Ser Xaa Phe Thr Gly 100 105 110 Arg 39 459 DNA Oryza sativa 39 cgctgccccg tcaccacgct ctgcatcggc caggccgcgt ccatgggctc cctcctgctc 60 gccgccggcg cgcgcgggga gcgccgggcg ctgcccaacg cgcgggtcat gattcaccag 120 ccatccgggg gcgcgcaggg ccaggccacc gacatcgcca tccaggccaa ggagattctc 180 aagctgcgcg accgcctcaa caagatctac cagaagcaca ccggccagga gatcgacaag 240 atcgagcagt gcatggagcg cgacctcttc atggaccccg aggaggcgcg cgattggggg 300 ctcatcgacg aggtaattga gaaccgcccc gcgtccctga tacccgaggg cgccactggc 360 gttgacctgc cgcaccacag cgccgctggc gtcggcggaa ggggcagaga tgtcgaggag 420 ccctccgcgg tgtgagctgt ggccgcaaag gtgaaacct 459 40 109 PRT Oryza sativa 40 Arg Cys Pro Val Thr Thr Leu Cys Ile Gly Gln Ala Ala Ser Met Gly 1 5 10 15 Ser Leu Leu Leu Ala Ala Gly Ala Arg Gly Glu Arg Arg Ala Leu Pro 20 25 30 Asn Ala Arg Val Met Ile His Gln Pro Ser Gly Gly Ala Gln Gly Gln 35 40 45 Ala Thr Asp Ile Ala Ile Gln Ala Lys Glu Ile Leu Lys Leu Arg Asp 50 55 60 Arg Leu Asn Lys Ile Tyr Gln Lys His Thr Gly Gln Glu Ile Asp Lys 65 70 75 80 Ile Glu Gln Cys Met Glu Arg Asp Leu Phe Met Asp Pro Glu Glu Ala 85 90 95 Arg Asp Trp Gly Leu Ile Asp Glu Val Ile Glu Asn Arg 100 105 41 466 DNA Glycine max 41 ggagcgtttc cagagtgtta taagtcagct tttccaatac aggataatcc gttgtggtgg 60 agcagttgat gacgatatgg caaacatcat agttgctcag ctcctgtacc tcgacgctgt 120 tgatcctaac aaggatattg tcatgtatgt aaattctcca ggagggtcgg ttacagctgg 180 aatggctata tttgatacaa tgaggcatat ccgacctgat gtgtctactg tttgtgttgg 240 attagcagct agtatgggag cttttctgct gagcgcaggg acaaaaggaa agagatacag 300 cttgccaaat tcaaggataa tgattcatca accgcttggt ggtgctcaag gagggcaaac 360 tgacatagat attcaggcta atgaaatgct gcatcaaaag gcaaatctga atggatatct 420 cgcctatcac actggccaaa gtttagacaa agatcaacca agatac 466 42 150 PRT Glycine max 42 Glu Arg Phe Gln Ser Val Ile Ser Gln Leu Phe Gln Tyr Arg Ile Ile 1 5 10 15 Arg Cys Gly Gly Ala Val Asp Asp Asp Met Ala Asn Ile Ile Val Ala 20 25 30 Gln Leu Leu Tyr Leu Asp Ala Val Asp Pro Asn Lys Asp Ile Val Met 35 40 45 Tyr Val Asn Ser Pro Gly Gly Ser Val Thr Ala Gly Met Ala Ile Phe 50 55 60 Asp Thr Met Arg His Ile Arg Pro Asp Val Ser Thr Val Cys Val Gly 65 70 75 80 Leu Ala Ala Ser Met Gly Ala Phe Leu Leu Ser Ala Gly Thr Lys Gly 85 90 95 Lys Arg Tyr Ser Leu Pro Asn Ser Arg Ile Met Ile His Gln Pro Leu 100 105 110 Gly Gly Ala Gln Gly Gly Gln Thr Asp Ile Asp Ile Gln Ala Asn Glu 115 120 125 Met Leu His Gln Lys Ala Asn Leu Asn Gly Tyr Leu Ala Tyr His Thr 130 135 140 Gly Gln Ser Leu Asp Lys 145 150 43 617 DNA Triticum aestivum unsure (358) unsure (402) unsure (410) unsure (439) unsure (447) unsure (495) unsure (571) unsure (574) unsure (600) unsure (602) 43 ggcggtcctg tggaggatga tatggccaac gtcattgttg cgcagctgct atacctggac 60 gccgttgatc ctaacaagga tatcattatg tatgtgaact ctccaggagg atcagtgaca 120 gctgggatgg ccatatttga tacaatgaag catatcaggc ctgatgtttc gacagtttgt 180 atcggacttg ctgcaagtat gggtgctttt ctacttagcg gtgggacgaa agggaagagg 240 tacagcttac ctaactcaag aataatgatc catcagcctc ttgggaggag cccaaggaca 300 agagaccgac cttgagattc caaggccaaa tgagatgctg caccacaagg ccaacttnta 360 acggatacct agcataccac actgggcagc ccctggataa gncaatgtan atactgaccg 420 tgacttcctc aagagcgcna aaggagnaaa ggagtatggg ccttattgat ggagtaatcg 480 tgaaccctct taaancgctg caaccactcc agctccagtt agccatccgt gcacaaaatc 540 tatgccgctc aagcaatttt gtgtgatctc nganttgtgt tgtacacctg ttttcgtagn 600 cngctaaatg ctttgat 617 44 95 PRT Triticum aestivum 44 Gly Gly Pro Val Glu Asp Asp Met Ala Asn Val Ile Val Ala Gln Leu 1 5 10 15 Leu Tyr Leu Asp Ala Val Asp Pro Asn Lys Asp Ile Ile Met Tyr Val 20 25 30 Asn Ser Pro Gly Gly Ser Val Thr Ala Gly Met Ala Ile Phe Asp Thr 35 40 45 Met Lys His Ile Arg Pro Asp Val Ser Thr Val Cys Ile Gly Leu Ala 50 55 60 Ala Ser Met Gly Ala Phe Leu Leu Ser Gly Gly Thr Lys Gly Lys Arg 65 70 75 80 Tyr Ser Leu Pro Asn Ser Arg Ile Met Ile His Gln Pro Leu Gly 85 90 95 45 521 DNA Triticum aestivum unsure (384) unsure (469) 45 ctctacatca actcccccgg gggcgtcgtc accgccgggc tcgccatcta cgacaccatg 60 cagtacatcc gctgccccgt caacaccatc tgcatcggcc aggccgcctc catgggctcc 120 ctcctcctcg ccgccggcgc gcgcggggag aggcgggcgc tgcccaacgc cagggtcatg 180 atccaccagc cctccggcgg ggcccagggc caggccaccg acatcgccat ccaggccaag 240 gagatactca aagctgcgcg accgcctcaa caagatctac gccaagcaca cgggccaaga 300 acatcgacaa gatcgagcag tgcatggagc gtgacctttt catggacccc cgaggaggcc 360 gcgaatgggg ggtttataga cgangtcatc gagaacgccc ggctccctca tcctgatggc 420 tcatgccgtt gaccgcctca cacggtgggg gccccgcgcc aacggcgtng caaggaaagg 480 atatggagga cctccgcgta taagggtggc aagcacaaag g 521 46 84 PRT Triticum aestivum 46 Leu Tyr Ile Asn Ser Pro Gly Gly Val Val Thr Ala Gly Leu Ala Ile 1 5 10 15 Tyr Asp Thr Met Gln Tyr Ile Arg Cys Pro Val Asn Thr Ile Cys Ile 20 25 30 Gly Gln Ala Ala Ser Met Gly Ser Leu Leu Leu Ala Ala Gly Ala Arg 35 40 45 Gly Glu Arg Arg Ala Leu Pro Asn Ala Arg Val Met Ile His Gln Pro 50 55 60 Ser Gly Gly Ala Gln Gly Gln Ala Thr Asp Ile Ala Ile Gln Ala Lys 65 70 75 80 Glu Ile Leu Lys 47 900 DNA Zea mays 47 ccacgcgtcc gagctcctcc tccttgacgc catcgacccg gactctgaca tccgcctctt 60 cgtcaactca ccagggggat cccttagcgc aacaatggcc atctatgatg taatgcagct 120 tgtgagggca gacgtgtcca ctattggaat gggcatagct ggatcaacag cttctataat 180 ccttggtggt ggcacgaagg gcaagcgatt tgccatgccc aacaccagga ttatgatcca 240 tcagcctgtc ggaggtgcaa gcgggcaggc cctagatgta gaggtccaag cgaaggagat 300 attgaccaac aagaggaatg tcattcggat cgtatcaggc ttcacaggcc gcactcctga 360 gcaggtagag aaagacattg acagagatcg ttacatgggc cctctcgagg ctgtcgatta 420 tggactcatt gatggcgtga tcgatggaga cagtattatc ccacttgagc ctgtcccgga 480 gagggtgaag cctaagtaca actacgaaga gctgtacaag gatccacaga agtttcttac 540 accagatgtc ccagatgatg agatatacta gtcgaaaagt tgtattttgt gcgaatgtta 600 agtctgttct tcagcaagca gatgtttttc gtcgcttgta gctgtcaaac caaccatagc 660 actagtagct tattgatctt gtttactgac tggatggtga ttcgagcagg caactagaac 720 ctgttggttg tgtttctggt gttacattgt ggtgttagaa tggtccggct gtttcgtttt 780 gaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 840 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 900 48 189 PRT Zea mays 48 His Ala Ser Glu Leu Leu Leu Leu Asp Ala Ile Asp Pro Asp Ser Asp 1 5 10 15 Ile Arg Leu Phe Val Asn Ser Pro Gly Gly Ser Leu Ser Ala Thr Met 20 25 30 Ala Ile Tyr Asp Val Met Gln Leu Val Arg Ala Asp Val Ser Thr Ile 35 40 45 Gly Met Gly Ile Ala Gly Ser Thr Ala Ser Ile Ile Leu Gly Gly Gly 50 55 60 Thr Lys Gly Lys Arg Phe Ala Met Pro Asn Thr Arg Ile Met Ile His 65 70 75 80 Gln Pro Val Gly Gly Ala Ser Gly Gln Ala Leu Asp Val Glu Val Gln 85 90 95 Ala Lys Glu Ile Leu Thr Asn Lys Arg Asn Val Ile Arg Ile Val Ser 100 105 110 Gly Phe Thr Gly Arg Thr Pro Glu Gln Val Glu Lys Asp Ile Asp Arg 115 120 125 Asp Arg Tyr Met Gly Pro Leu Glu Ala Val Asp Tyr Gly Leu Ile Asp 130 135 140 Gly Val Ile Asp Gly Asp Ser Ile Ile Pro Leu Glu Pro Val Pro Glu 145 150 155 160 Arg Val Lys Pro Lys Tyr Asn Tyr Glu Glu Leu Tyr Lys Asp Pro Gln 165 170 175 Lys Phe Leu Thr Pro Asp Val Pro Asp Asp Glu Ile Tyr 180 185 49 690 DNA Oryza sativa 49 cgctgccccg tcaccacgct ctgcatcggc caggccgcgt ccatgggctc cctcctgctc 60 gccgccggcg cgcgcgggga gcgccgggcg ctgcccaacg cgcgggtcat gattcaccag 120 ccatccgggg gcgcgcaggg ccaggccacc gacatcgcca tccaggccaa ggagattctc 180 aagctgcgcg accgcctcaa caagatctac cagaagcaca ccggccagga gatcgacaag 240 atcgagcagt gcatggagcg cgacctcttc atggaccccg aggaggcgcg cgattggggg 300 ctcatcgacg aggtaattga gaaccgcccc gcgtccctga tacccgaggg cgccactggc 360 gttgacctgc cgcaccacag cgccgctggc gtcggcggaa ggggcagaga tgtcgaggag 420 ccctccgcgg tgtgagctgt ggccgcaaag gtgaaacctt ttcgtgtccc atggccatgt 480 tgttgttgtt attagatcca aggttcagtt cttatactac ataaacttaa cttgttatta 540 ttcaggttgc cacttgttat tcaggttgcc gatgtgttcg gctccttaca tgttgtcttg 600 attgcctgaa ttgagctact gctgatattt attgcaaatc taaggaaatt ttattccttc 660 catactgata aaaaaaaaaa aaaaaaaaaa 690 50 144 PRT Oryza sativa 50 Arg Cys Pro Val Thr Thr Leu Cys Ile Gly Gln Ala Ala Ser Met Gly 1 5 10 15 Ser Leu Leu Leu Ala Ala Gly Ala Arg Gly Glu Arg Arg Ala Leu Pro 20 25 30 Asn Ala Arg Val Met Ile His Gln Pro Ser Gly Gly Ala Gln Gly Gln 35 40 45 Ala Thr Asp Ile Ala Ile Gln Ala Lys Glu Ile Leu Lys Leu Arg Asp 50 55 60 Arg Leu Asn Lys Ile Tyr Gln Lys His Thr Gly Gln Glu Ile Asp Lys 65 70 75 80 Ile Glu Gln Cys Met Glu Arg Asp Leu Phe Met Asp Pro Glu Glu Ala 85 90 95 Arg Asp Trp Gly Leu Ile Asp Glu Val Ile Glu Asn Arg Pro Ala Ser 100 105 110 Leu Ile Pro Glu Gly Ala Thr Gly Val Asp Leu Pro His His Ser Ala 115 120 125 Ala Gly Val Gly Gly Arg Gly Arg Asp Val Glu Glu Pro Ser Ala Val 130 135 140 51 874 DNA Glycine max 51 gcacgaggga gcgtttccag agtgttataa gtcagctttt ccaatacagg ataatccgtt 60 gtggtggagc agttgatgac gatatggcaa acatcatagt tgctcagctc ctgtacctcg 120 acgctgttga tcctaacaag gatattgtca tgtatgtaaa ttctccagga gggtcggtta 180 cagctggaat ggctatattt gatacaatga ggcatatccg acctgatgtg tctactgttt 240 gtgttggatt agcagctagt atgggagctt ttctgctgag cgcagggaca aaaggaaaga 300 gatacagctt gccaaattca aggataatga ttcatcaacc gcttggtggt gctcaaggag 360 ggcaaactga catagatatt caggctaatg aaatgctgca tcataaggca aatctgaatg 420 gatatctcgc ctatcacact ggccaaagtt tagacaagat caaccaggat acagaccgtg 480 actttttcat gagtgcaaaa gaagccaagg aatatggact catagatggt gtcattatga 540 atcctctcaa agctctccag ccattagagg ctgcagcaga aggtaaagac cgggctagtg 600 tttgaacatg agaatgttgc actttaattt ccaaggtata aaaaatcata gtgttagact 660 gtaagatgtt tttggttgct gagtccaact taattttttt ttacggatgt tgatacctgt 720 gcccatgtac caaaaatgag gcgaaattga tactatttat ttaatattca ctgcttcaga 780 gtttatactg acagaaggtt ctttaatgga acctgaatgt gattttaact tcaagcattc 840 ttttgtgatg aactgaaaaa aaaaaaaaaa aaaa 874 52 200 PRT Glycine max 52 Thr Arg Glu Arg Phe Gln Ser Val Ile Ser Gln Leu Phe Gln Tyr Arg 1 5 10 15 Ile Ile Arg Cys Gly Gly Ala Val Asp Asp Asp Met Ala Asn Ile Ile 20 25 30 Val Ala Gln Leu Leu Tyr Leu Asp Ala Val Asp Pro Asn Lys Asp Ile 35 40 45 Val Met Tyr Val Asn Ser Pro Gly Gly Ser Val Thr Ala Gly Met Ala 50 55 60 Ile Phe Asp Thr Met Arg His Ile Arg Pro Asp Val Ser Thr Val Cys 65 70 75 80 Val Gly Leu Ala Ala Ser Met Gly Ala Phe Leu Leu Ser Ala Gly Thr 85 90 95 Lys Gly Lys Arg Tyr Ser Leu Pro Asn Ser Arg Ile Met Ile His Gln 100 105 110 Pro Leu Gly Gly Ala Gln Gly Gly Gln Thr Asp Ile Asp Ile Gln Ala 115 120 125 Asn Glu Met Leu His His Lys Ala Asn Leu Asn Gly Tyr Leu Ala Tyr 130 135 140 His Thr Gly Gln Ser Leu Asp Lys Ile Asn Gln Asp Thr Asp Arg Asp 145 150 155 160 Phe Phe Met Ser Ala Lys Glu Ala Lys Glu Tyr Gly Leu Ile Asp Gly 165 170 175 Val Ile Met Asn Pro Leu Lys Ala Leu Gln Pro Leu Glu Ala Ala Ala 180 185 190 Glu Gly Lys Asp Arg Ala Ser Val 195 200 53 755 DNA Triticum aestivum 53 gcacgagggc ggtcctgtgg aggatgatat ggccaacgtc attgttgcgc agctgctata 60 cctggacgcc gttgatccta acaaggatat cattatgtat gtgaactctc caggaggatc 120 agtgacagct gggatggcca tatttgatac aatgaagcat atcaggcctg atgtttcgac 180 agtttgtatc ggacttgctg caagtatggg tgcttttcta cttagcggtg ggacgaaagg 240 gaagaggtac agcttaccta actcaagaat aatgatccat cagcctcttg gaggagccca 300 aggacaagag accgaccttg agatccaggc caatgagatg ctgcaccaca aggccaactt 360 gaacggatac ctagcatacc acactgggca gcccctggat aagatcaatg tagatactga 420 ccgtgacttc ttcatgagcg cgaaggaggc aaaggagtat ggccttattg atggagtaat 480 cgtgaaccct cttaaagcgc tgcaaccact tccagcttcc agttagccat gccgtgcaca 540 aaatctatgc cgctccaagc atttttgttg tgatcttctg gagttgtgtt tgtaccacgc 600 tgttttcgtt agtctggcta gatgcttttg taatttcacg ttctgaagct ttcacaggtt 660 gtacggaaca gatgcactac tagaatgttc atcgtttgcg gtaagatgtt tgcacgtgag 720 tcgacgttgt ttttgttaaa aaaaaaaaaa aaaaa 755 54 174 PRT Triticum aestivum 54 His Glu Gly Gly Pro Val Glu Asp Asp Met Ala Asn Val Ile Val Ala 1 5 10 15 Gln Leu Leu Tyr Leu Asp Ala Val Asp Pro Asn Lys Asp Ile Ile Met 20 25 30 Tyr Val Asn Ser Pro Gly Gly Ser Val Thr Ala Gly Met Ala Ile Phe 35 40 45 Asp Thr Met Lys His Ile Arg Pro Asp Val Ser Thr Val Cys Ile Gly 50 55 60 Leu Ala Ala Ser Met Gly Ala Phe Leu Leu Ser Gly Gly Thr Lys Gly 65 70 75 80 Lys Arg Tyr Ser Leu Pro Asn Ser Arg Ile Met Ile His Gln Pro Leu 85 90 95 Gly Gly Ala Gln Gly Gln Glu Thr Asp Leu Glu Ile Gln Ala Asn Glu 100 105 110 Met Leu His His Lys Ala Asn Leu Asn Gly Tyr Leu Ala Tyr His Thr 115 120 125 Gly Gln Pro Leu Asp Lys Ile Asn Val Asp Thr Asp Arg Asp Phe Phe 130 135 140 Met Ser Ala Lys Glu Ala Lys Glu Tyr Gly Leu Ile Asp Gly Val Ile 145 150 155 160 Val Asn Pro Leu Lys Ala Leu Gln Pro Leu Pro Ala Ser Ser 165 170 55 788 DNA Triticum aestivum 55 ccatcagcct ctacatcaac tcccccgggg gcgtcgtcac cgccgggctc gccatctacg 60 acaccatgca gtacatccgc tgccccgtca acaccatctg catcggccag gccgcctcca 120 tgggctccct cctcctcgcc gccggcgcgc gcggggagag gcgggcgctg cccaacgcca 180 gggtcatgat ccaccagccc tccggcgggg cccagggcca ggccaccgac atcgccatcc 240 aggccaagga gatactcaag ctgcgcgacc gcctcaacaa gatctacgcc aagcacacgg 300 gccagaacat cgacaagatc gagcagtgca tggagcgtga ccttttcatg gaccccgagg 360 aggcccgcga atgggggctt atagacgagg tcatcgagaa ccgcccggcc tccctcatgc 420 ctgatggcct cagtgccgtt gacccgcctc accacggtgg gggcgccggc gccaacggcc 480 gtggcaggga cagggatatg gaggagccct ccgcggtatg aggggtggcc aggccacaaa 540 ggtgaaacct ttttctgagt ccggtggcta tgttgtttgt tgttagatct aagttttgat 600 tcctaataca acaggtcaac ttggtatcct cttcctgttg tttcaattgc ctgaactgag 660 ctattgccga tatttattgc aactcgtaaa aaggaatttc gttcctttga tactgataaa 720 ttgatagtgt ggtgaatatc agttatacga tcaatttcaa gtcacagcaa aaaaaaaaaa 780 aaaaaaaa 788 56 172 PRT Triticum aestivum 56 Ile Ser Leu Tyr Ile Asn Ser Pro Gly Gly Val Val Thr Ala Gly Leu 1 5 10 15 Ala Ile Tyr Asp Thr Met Gln Tyr Ile Arg Cys Pro Val Asn Thr Ile 20 25 30 Cys Ile Gly Gln Ala Ala Ser Met Gly Ser Leu Leu Leu Ala Ala Gly 35 40 45 Ala Arg Gly Glu Arg Arg Ala Leu Pro Asn Ala Arg Val Met Ile His 50 55 60 Gln Pro Ser Gly Gly Ala Gln Gly Gln Ala Thr Asp Ile Ala Ile Gln 65 70 75 80 Ala Lys Glu Ile Leu Lys Leu Arg Asp Arg Leu Asn Lys Ile Tyr Ala 85 90 95 Lys His Thr Gly Gln Asn Ile Asp Lys Ile Glu Gln Cys Met Glu Arg 100 105 110 Asp Leu Phe Met Asp Pro Glu Glu Ala Arg Glu Trp Gly Leu Ile Asp 115 120 125 Glu Val Ile Glu Asn Arg Pro Ala Ser Leu Met Pro Asp Gly Leu Ser 130 135 140 Ala Val Asp Pro Pro His His Gly Gly Gly Ala Gly Ala Asn Gly Arg 145 150 155 160 Gly Arg Asp Arg Asp Met Glu Glu Pro Ser Ala Val 165 170 57 1592 DNA Zea mays 57 gcacgaggaa accagcattt gctactagta gaaagcaaaa cgagctttgg gtatccattc 60 ttgagaaggc ttatgcaaaa cttcatggct cttatgaggc attggaaggt gggcttgttc 120 aagatgctct agtcgatctc acaggaggag ctggtgaaga gattgatatg cgaagtcctc 180 aagcccaact tgatcttgct agtggaagat tgtggtcgca gttgttgcat ttcaaacaag 240 aaggttttct tcttggtgct ggaagtcctt ctggatctga tgctcacatc tcatcaagtg 300 gcattgttca gggacatgcg tactcaattt tgcaggtaag agaagttgat ggccacaaac 360 tcatccaaat cagaaatcca tgggcaaatg aagttgaatg gaatggacca tggtcagact 420 cgtcaccaga gtggacggaa cggatgaagc ataagctcat gcatgttcca cagtcgaaga 480 atggggtatt ctggatgtct tggcaagatt ttcagattca ctttcggtca atatatgttt 540 gtcgtgttta tccacctgag atgcgttact ctgtccatgg gcaatggcgt ggctacaatg 600 caggtggttg ccaagattat gactcgtggc accaaaatcc acagtatcga cttagagtaa 660 caggacgtga tgcactatac cctgttcacg tttttattac ccttactcag ggtgttggtt 720 tctctagaaa gacgaatggt tttcggaact accaatctag ccatgattct tcaatgtttt 780 acattggaat gaggatactc aagacacagg gctgccgtgc tgcttacaat atctacatgc 840 atgaaagcgc tggtggaaca gattacgtta actcgaggga gatatcatgc gaactggtct 900 tggatcctta tcccaaaggg tacacaattg tgccaactac catccaccct ggggaggaag 960 caccttttgt tttgtcagtt ttttcaaaag catcaatcag actagaggct gtttagttca 1020 agattgagat cccatgtgtt tgatggtagc tgcgtctgct gggcacccgt gcacgcagga 1080 tccagctgtg ggttctcggg aactagataa tgggtatagg aattgcctcc tggacaactt 1140 caatcaatct tgctgcatgc aagtacctaa gttcggttgc ttgttgcaga tctgacaaac 1200 ggcaatgctt cttgtgctga agggaaagga gagaaggcat gatccatggt tctttggtag 1260 ctgcgcaaag tgcagggtga gaggcttggt tcaatgtttg tagatagccg tggtaactga 1320 cctggtagcc catcctatgt ataggtgtcc cgtttaccct gtaaatgcta tagagttagg 1380 ttaggtagcc tgtcgttcct gttaacgcat agggctctta tgcagctgtg aaatgtcttg 1440 ttggcaagct gcagttttgc tgatttgagc gtggagtagt cggccatagc tgttcccatt 1500 ggtttgccct gtatgtaatc ggaatctgat gtcattcaat gaacctattt tttgggtgcc 1560 atgcgaagct gtctaaaaaa aaaaaaaaaa aa 1592 58 540 DNA Triticum aestivum unsure (26) unsure (46) unsure (79) unsure (107) 58 aaagtctcga ctgcgaattt gataancttc tttaggttga aggagncatt gcataagcgt 60 gtcatcgccc aagacgagnc tgtgaaagca ataagtcggt ctgttcnccg tgctcgtgtg 120 gccctcaaga gccccaacag accaattgcg agcttcattt ttgcaggtcc cactggtgtt 180 gggaaatcag agcttgcaaa gactctggca tcatattact tcggctctga agaagccatg 240 atccggctgg atatgagtga gttcatggag aggcacactg tgtccaagtt gatcggttca 300 ccaccaggct atgtgggata cactgaaggt gggcagctga cggaggcggt tcgacggcgg 360 ccatacagcg ttgtgctttt tgatgagatt gagaaggcac atccggatgt gttcaacatg 420 atgctccaga tcctggaaga cggaaggtta accgacagca aggggagaac agtggacttc 480 aagaacacgc tcctgataat gacatcaaat gttgggagca gcgtgattga gaagaagaag 540 

What is claimed is:
 1. An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 40 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 30, 32, and 34, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
 2. An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 150 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 10, 36, and 38, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
 3. An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 200 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12 and 40, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
 4. An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide of at least 175 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 16, 18, 42, 44, and 46, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
 5. An isolated polynucleotide comprising a first nucleotide sequence encoding a polypeptide selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 48, 50, 52, 54, and 56, or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
 6. The isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5, wherein the first nucleotide sequence consists of a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and
 56. 7. The isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5 wherein the nucleotide sequences are DNA.
 8. The isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5 wherein the nucleotide sequences are RNA.
 9. A chimeric gene comprising the isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5 operably linked to suitable regulatory sequences.
 10. An isolated host cell comprising the chimeric gene of claim
 9. 11. A host cell comprising an isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim
 5. 12. The host cell of claim 11 wherein the host cell is selected from the group consisting of yeast, bacteria, plant, and virus.
 13. A virus comprising the isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim
 5. 14. A polypeptide of at least 40 amino acids that has at least 85% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 30, 32, and
 34. 15. A polypeptide of at least 150 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:8, 10, 36, and
 38. 16. A polypeptide of at least 200 amino acids that has at least 80% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:12 and
 40. 17. A polypeptide of at least 175 amino acids that has at least 95% identity based on the Clustal method of alignment when compared to a polypeptide selected from the group consisting of SEQ ID NOs:14, 16, 18, 42, 44, and
 46. 18. A polypeptide selected from the group consisting of SEQ ID NOs:20, 22, 24, 26, 28, 48, 50, 52, 54, and
 56. 19. A method of selecting an isolated polynucleotide that affects the level of expression of a proteinase polypeptide in a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from an isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5; (b) introducing the isolated polynucleotide into a plant cell; (c) measuring the level of a polypeptide in the plant cell containing the polynucleotide; and (d) comparing the level of polypeptide in the plant cell containing the isolated polynucleotide with the level of polypeptide in a plant cell that does not contain the isolated polynucleotide.
 20. The method of claim 19 wherein the isolated polynucleotide consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 that codes for the polypeptide selected from the group consisting of SEQ ID NOs:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, and
 56. 21. A method of selecting an isolated polynucleotide that affects the level of expression of a proteinase polypeptide in a plant cell, the method comprising the steps of: (a) constructing an isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5; (b) introducing the isolated polynucleotide into a plant cell; (c) measuring the level of polypeptide in the plant cell containing the polynucleotide; and (d) comparing the level of polypeptide in the plant cell containing the isolated polynucleotide with the level of polypeptide in a plant cell that does not contain the polynucleotide.
 22. A method of obtaining a nucleic acid fragment encoding a proteinase polypeptide comprising the steps of: (a) synthesizing an oligonucleotide primer comprising a nucleotide sequence of at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences; and (b) amplifying a nucleic acid sequence using the oligonucleotide primer.
 23. A method of obtaining a nucleic acid fragment encoding a proteinase polypeptide comprising the steps of: (a) probing a cDNA or genomic library with an isolated polynucleotide comprising at least one of 30 contiguous nucleotides derived from a nucleotide sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such nucleotide sequences; (b) identifying a DNA clone that hybridizes with the isolated polynucleotide; (c) isolating the identified DNA clone; and (d) sequencing the cDNA or genomic fragment that comprises the isolated DNA clone.
 24. A composition comprising the isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim
 5. 25. A composition comprising the isolated polynucleotide of claim 14, claim 15, claim 16, claim 17, or claim
 18. 26. An isolated polynucleotide comprising the nucleotide sequence having at least one of 30 contiguous nucleotides derived from a nucleic acid sequence selected from the group consisting of SEQ ID NOs:1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, and 55 and the complement of such sequences.
 27. An expression cassette comprising an isolated polynucleotide of claim 1, claim 2, claim 3, claim 4, or claim 5 operably linked to a promoter.
 28. A method for positive selection of a transformed cell comprising: (a) transforming a host cell with the chimeric gene of claim 9 or an expression cassette of claim 27; and (b) growing the transformed host cell under conditions which allow expression of the polynucleotide.
 29. The method of claim 28 wherein the plant cell is a monocot.
 30. The method of claim 28 wherein the plant cell is a dicot. 